Document Type : Original Research Article
Subjects
Introduction
Considerable evidence on the expansion of the geographical range and the incidence of dangerous fungal diseases around the world have emerged in recent years. Various reasons have been cited for the increased prevalence of dangerous fungi, including global warming, increased communication, and the expansion of global trade (1). In recent years, the occurrence of invasive and deadly fungal infections has significantly increased among COVID-19 patients (2). Due to the widespread use of antifungal drugs for common fungal infections such as oral and vaginal Candida or respiratory fungi such as Aspergillus, the risks of more aggressive forms of infections in the general population are always increasing. Using substances that inhibit the growth of fungi in different environments is one of the most common ways to prevent infection.
Curcumin is a natural phenol with antimicrobial characteristics against a broad spectrum of pathogens (3). This compound is hydrophobic and is not bioavailable systemically. Curcumin nanoformulations have drawn attention as a solution for this drawback because they can increase curcumin hydrophilicity and boost its bioavailability (4). Recent studies have advanced antifungal nanomaterials, demonstrating the efficacy of metal-ligand complexes in combating drug-resistant fungi through enhanced bioavailability and targeted activity (5, 6). Phytochemical-based formulations, including those inspired by curcumin, offer promising antimicrobial properties but highlight the need for eco-friendly synthesis to improve stability and reduce toxicity (7).
Apart from various medicinal properties, curcumin, as an active phytochemical compound, can be applied for the chemical synthesis of metal nanoparticles. Metal nanoparticles have widely entered in medical research (8), diagnostics (9), cancer treatments (10), health industry (11), and antimicrobial applications (12). Here the aqueous solution of Cur@AuNPs through a one pot green synthesis procedure without any other chemicals or solvent. The antifungal effect of the synthesized nanoparticles has been investigated for the first time against various strains of Candida and Aspergillus fungus.
Briefly, 20 mM solution of curcumin (diferuloylmethane, C21H20O6 65%, Sigma-Aldrich, USA) in DMSO (C2H6OS, 99.5%, Gibco, Life Technologies GmbH, Karlsruhe, Germany) was prepared. A round bottom flask was filled with 15 mL of deionized water (DIW). The water’s pH was adjusted to approximately 9 using a 150 mM potassium carbonate (K₂CO₃) solution sourced from Sigma-Aldrich, USA. Subsequently, 100 µL of curcumin solution was added dropwise into the stirred DIW, causing the initially yellow curcumin solution to shift to a red hue. After a three-minute interval, 2.5 mL of tetrachloroauric (III) acid trihydrate (HAuCl₄·3H₂O, 2.5 mM, Sigma-Aldrich, Germany) was slowly introduced into the curcumin mixture. The reaction was maintained under continuous stirring for three hours, then the mixture was kept undisturbed in the dark for a period of three days. (13, 14) According to our previous study, the nanoparticles were washed through a series of centrifugation and decantation to remove unreacted reagents (15, 16).
Characterizations of the Cur@AuNPs
Prior to conducting any characterization, the produced Cur@AuNPs were filtered using a 0.2 µm polyvinylidene fluoride (PVDF) membrane. Transmission electron microscopy (Zeiss EM 900, Germany) was employed to examine the size and structural features of the nanoparticles. The nanoparticles’ optical characteristics were evaluated via UV-Visible spectroscopy, utilizing a double-beam UV-Vis absorption spectrophotometer (SPEKOL 2000, Analytik Jena, UK) equipped with a quartz cuvette having a 1 cm optical path length. Additionally, dynamic light scattering (DLS) measurements to determine hydrodynamic size were performed using the NANO-flex Particle Sizer (Germany), while the surface charge (zeta potential) was assessed with a Zeta-check instrument (Microtrac, Germany). Finally, the concentration of gold ions within the nanoparticle suspensions was quantified through inductively coupled plasma optical emission spectroscopy (ICP-OES).
In vitro cytotoxicity analysis
HepG2 cells were obtained from the Pasteur Institute of Iran, Tehran. Cells were cultured in DMEM culture medium supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution. The cells were kept in an atmosphere of 5% CO2 air at 37°C and the culture medium was changed daily. To investigate the cytotoxicity of Cur@AuNPs, cells were seeded at approximately 100,000 cells per well of a 96-well plate, and after the cells were attached to the plate, the cells were treated with culture containing different concentrations of nanoparticles and incubated for 24 hours. Then the cells were washed with PBS and incubated with 100 µL of MTT solution in buffer or culture medium at a concentration of 0.5 mg/mL for 2-4 hours. Following incubation, the MTT solution was removed and replaced with 100 µL of DMSO. After gently shaking the plate for 2 minutes, absorbance was measured at 570 nm using a microplate reader.
Antifungal activity testing of Cur@AuNPs
A set of 30 clinically important fungal strains including Candida albicans (n=6), Candida parapsilosis (n=6), Candida krusei (n=6), Aspergillus fumigatus (n=6), and Aspergillus flavus (n=6) were investigated in the current study. These fungi have previously been identified using sequence analysis of ITS1-5.8S rDNA-ITS2 (for Candida species) or beta-tubulin (for Aspergillus species) markers.
The antifungal activity of Cur@AuNPs was determined according to the Clinical and Laboratory Standards Institute (CLSI) protocols for yeasts and filamentous fungi (17). The susceptibility of the isolates to itraconazole (Behvazan Co., Iran) and fluconazole (Amin Pharma. Co., Iran) was also determined using the same method. The tested concentrations for Cur@AuNPs, itraconazle, and fluconazole were 1–512 µg/mL, 0.03–16 µg/mL, and 0.125–64 µg/mL, respectively. C. parapsilosis ATCC 22019 and C. krusei ATCC 5268 were used as quality control. The value of minimum inhibitory concentration (MIC) was considered as the lowest concentration of the nanoparticle that was able to make 50% inhibition (100% for itraconazole against Aspergillus species) in comparison to drug-free growth control wells. Minimum fungicidal concentration (MFC) was determined based on a method described previously (18).
Specimen preparation for electron microscopy
For analysis of the Cur@AuNPs interaction with C. albicans, cultures were incubated with Cur@AuNPs at 32 µg/mL concentration for 24 hours. After nanoparticle incubation, the treated microorganisms were washed with PBS carefully. For primary fixation a 2-hour incubation with 2.5% glutaraldehyde was applied. Free glutaraldehyde was removed through a series of washing procedures with PBS. Secondary fixation was carried out by 1.5-hour incubation with 1% osmium tetroxide. Dehydration of the fungus was performed in acetone (50, 70, 90, 100%), then infiltrated by resin and finally embedded in pure resin (Epon 812, TAAB, UK). then, the ultra-microtome was applied to obtain thin sections of 50 nm. Finally, the provided slices were stained with uranyl acetate and lead citrate for investigation under TEM (Zeiss EM 900, Germany)
The data are expressed as means ± SD. Differences between groups were assessed by one-way ANOVA. The diagram was analyzed and presented using Origin 6 software. The size distribution of the particles was obtained from the Gaussian fit of the diameter of 100 nanoparticles that have been analyzed with Digital Micrograph software..
Synthesis and characterizations
This green approach, leveraging curcumin’s dual role as reductant and stabilizer, yielded stable, spherical NPs, contrasting with slower kinetics in microbial green syntheses that often yield polydisperse particles (19) or an immunogenic response similar to previous experiences for gene delivery applications (20).
The formation of Cur@AuNPs was verified by the distinct plasmonic red color typical of gold nanoparticles (21). The observation that the plasmonic peak remains at essentially the same wavelength (524 nm vs. 525 nm) over a period of more than two months is a strong and direct indicator of high stability for the Cur@AuNPs. The UV-Vis spectra were applied to investigate the stability of the particles (Figure 1). Similar nanoparticles that have been synthesized by apigenin, which is a natural flavonoid, represent high stability in physiological environments (15). The very high stability of curcumin-coated silver nanoparticles was also investigated in our previous study (22). After making sure that the stable nanoparticles were synthesized, the hydrodynamic diameter of the particles was evaluated, which was 10.88±1.16 nm. TEM analysis revealed the size distribution and morphological characteristics of the synthesized Cur@AuNPs. Cur@AuNPs represent spherical morphology, with an average particle size of 7.5 ± 4.1 nm with all of the nanoparticles were almost spherical (Figure 2).
Studies have shown that nanoparticles produced via green synthesis often exhibit significant variability in both size and morphology (19). This heterogeneity arises because the reduction of metal ions by plant-derived compounds proceeds at a relatively slow rate, resulting in nanoparticles with a broad size distribution (23). In the preparation of Cur@AuNPs, curcumin must first be dissolved in DMSO and can then be further diluted with water, particularly at elevated pH levels. Raising the pH is crucial to facilitate the release of protons from curcumin’s hydroxyl groups, which aids in the reduction of gold ions. It is important to introduce HAuCl₄ within 5 minutes, as at pH 9, curcumin degradation intensifies beyond this time frame. By adding HAuCl4, primary gold nuclei are formed with electrons obtained from the degradation of curcumin. It has been demonstrated that small silver particles can have catalytic properties in the destruction of curcumin (24). The catalytic activity of gold was also declared (14). This can provide more electrons for the reduction of gold ions in the growth stage of the nuclei. The slower the growth of the particles, the more homogeneous the shape and size of the nanoparticles will be. That is why the growth of nanoparticles at room temperature without stirring for three days was chosen. To eliminate unbound curcumin, the Cur@AuNPs underwent multiple cycles of centrifugation followed by decantation. Studies have shown that performing this washing process four times effectively clears residual curcumin and gold ions (13, 14). The final nanoparticles had a negative charge of -27.9 ± 0.1 mV, which is actually due to the presence of the polyphenol coating caused by the polymerization of the broken curcuminoids on the surface of the nanoparticles.
A key challenge in utilizing metal nanoparticles for biomedical applications is their potential toxicity. Since gold is non-biodegradable, its long-term toxicity is a concern, with cytotoxicity depending on parameters such as size, shape, and surface chemistry. For instance, studies show that traditionally synthesized citrate-coated gold nanoparticles with the size of 5 nm are more toxic than 15 or 25 nm ones (25). The use of natural phytochemicals like curcumin for synthesis has produced nanoparticles with reduced toxicity. Curcumin-coated gold nanoparticles (Cur@AuNPs) have demonstrated high biocompatibility, showing no significant toxicity on human colorectal cancer HT29 (up to 50 μg/mL) (14), human monocytic cell line (THP-1) (26), human prostate cancer cell line (PC-3) VI paper (up to 280 µg/mL) (27), embryonic BD1X rat heart tissue (H9c2) (28), and peripheral blood mononuclear cells (PBMCs) (29). However, cytotoxic effects have been observed in other lines, such as L929 mouse fibroblastic cells (50% cell death at 120 μg/mL) (30), 3T3 Fibroblasts (31), human lung cancer (A549), human breast cancer (MDAMB-231), prostate cancer (DU145) cell lines (32), and MCF-7, as well as HCT-116 (33). In the present study on HepG2 cells, statistically significant effects begin at 4 μg/mL, yet over 90% of cells survive at 16 μg/mL (92.23%), and over 70% survive at 128 μg/mL (75.41%). By reviewing all these reports, it can be concluded that if Cur@AuNPs are washed well to remove ions, free curcumin, and other by-products not attached to the surface, the resulting nanoparticles are very biocompatible toward various cell lines.
Antifungal activity of Cur@AuNPs
MIC values of Cur@AuNPs, fluconazole, and itraconazole against Aspergillus and Candida isolates used in this study are shown in Table 1. All the Aspergillus isolates were unresponsive to fluconazole (geometric mean [GM] MIC: >64 µg/mL), while 5 and 4 isolates were susceptible and intermediate to itraconazole, respectively. Cur@AuNPs, similar to fluconazole, did not show any activity against the Aspergillus isolates (GM MIC: >128 µg/mL).
The lack of activity against Aspergillus's species may be attributed to several structural and mechanistic factors. Differences in fungal cell wall composition could play a key role, as Candida species are primarily composed of β-glucans and mannans, facilitating easier nanoparticle interaction, whereas Aspergillus features a higher chitin content and a more rigid structure that may impede Cur@AuNP penetration or adhesion (34). Additionally, variations in curcumin release kinetics or nanoparticle uptake might differ between the genera, with Aspergillus filamentous morphology potentially leading to reduced internalization compared to the yeast-like form of Candida (22). Furthermore, fungal efflux pumps and inherent drug resistance mechanisms in Aspergillus, such as those contributing to azole tolerance, could actively expel curcumin or limit Cur@AuNP efficacy (35). These observations align with studies on curcumin nanoformulations, which often show stronger antifungal effects against Candida than Aspergillus due to these biological barriers (4). Future experiments, including comparative uptake assays using fluorescently labeled nanoparticles, efflux pump inhibitor co-treatments, or cell wall disruption models, could further elucidate these mechanisms and guide optimizations for broader-spectrum activity.
The fungistatic effect of citrate-capped gold nanoparticles (36), and NaBH4-capped gold nanoparticles (37) was declared for Candida strains. Since the nanoparticles made with these chemicals do not have steric stability, they are very unstable in physiological environments. Therefore, they cannot be easily used for biomedical applications. These chemicals themselves have toxic effects on eukaryotic cells and their removal from the surface or solution of nanoparticles cannot be done easily (37). Even the comparative study of nanoparticles has shown that nanoparticles prepared with NaBH4 have more toxicity than the same nanoparticles that have been prepared by SnCl2 against Candida strains (37).
Regarding the Candida species, all but two (one C. albicans [MIC: 0.25 µg/mL] and one C. parapsilosis [MIC: 0.5 µg/mL] isolates), were resistant to fluconazole. There were also four isolates with MIC values of ≥16 µg/mL for itraconazole. The activity of Cur@AuNPs was much better against Candida species, with the superiority of C. albicans, compared to Aspergillus species. The GM MICs of Cur@AuNPs against C. albicans, C. parapsilosis, and C. krusei isolates were 25.40 µg/mL, 50.80 µg/mL, and 50.80 µg/mL, respectively. The inhibition activity of the curcumin against the Candida strains was attributed to hyphal development suppression by Cur (38). Also, the production of reactive oxygen species (ROS) from Cur (39), and gold nanoparticles has been reported (40). Regarding the MFC values, Cur@AuNPs did not demonstrate any fungicidal activity against Aspergillus and Candida species. The activity of itraconazole in this regard was much better than the fluconazole and Cur@AuNPs (Table 2).
Antifungal effects of gold nanoparticles with similar biogenic coatings on Aspergillus strains have been investigated in some studies. Such coatings for gold nanoparticles prepared by green chemistry method can be from all compounds of an extract of a substance such as a plant or mushroom, or a phytochemical isolated from an extract. Antifungal effects against the Aspergillus flavus strain have been observed for gold nanoparticles prepared with Agaricus bisporus mushroom extract. However, other strains of Aspergillus were resistant to the synthesized nanoparticles (41).
Antifungal effect toward Aspergillus flavus, Aspergillus niger, and Candida albicans for green synthesized gold nanoparticles from Abelmoschus esculentus extract was also demonstrated (42). Green-synthesized gold nanoparticles from Cannabis sativa represent an inhibitory effect against Aspergillus flavus, Aspergillus fumigatus, and Aspergillus niger (43). Quercetin is a natural flavonoid that could be applied for the synthesis of gold nanoparticles. The inhibitory effect of quercetin against A. fumigatus was reported by Milanezi et al (44). Cinnamaldehyde is the main flavonoid from the spice cinnamon and has been applied for the synthesis of gold nanoparticles. The synthesized Cinnamaldehyde-coated gold nanoparticles could inhibit the C. albicans at the 75 µg/ml concentration (45).
To evaluate the interaction between nanoparticles and fungal species, transmission electron microscopy imaging of the fungi treated with Cur@AuNPs was performed (Figure 4). The interaction of Cur@AuNPs with the Candida albicans samples could be classified into three categories based on the provided micrographs. It was observed that the Cur@AuNPs are accumulated on the surface of the cell wall/membrane (black arrows). This accumulation of nanoparticles on the cell wall can also have a negative effect on the process of cell mitosis. An example of a dividing cell is marked by a white line in the micrograph of Figure 4a. The interaction of metal nanoparticles on microorganisms’ membranes could also disturb the membrane potential and create pores. This phenomenon leads to disruption of the microorganism’s cell membrane/wall and the exit of ions and cytoplasmic fluid from the microorganism. The cell wall disruptor (orange arrows), and cytoplasmic shedding (green arrows) are observed in the presented TEM micrographs. The entrance of the Cur@AuNPs into the cytoplasm of the intact fungal cell is also clear in the TEM micrographs (Figure 4b-c).
The synthesis of biocompatible gold nanoparticles with the natural phenolic compound curcumin was performed through a single-step method in which curcumin acts both as a metal ion-reducing agent and as a stabilizing and coating agent. The synthesized Cur@AuNPs demonstrated high biocompatibility, with over 90% HepG2 cell viability observed at concentrations up to 16 µg/mL. Cur@AuNPs exhibited promising antifungal activity against various Candida species, including drug-resistant strains, but were ineffective against Aspergillus species. These results highlight the potential of Cur@AuNPs as alternative agents for Candida-related infections.
This study has been funded by Iran University of Medical Sciences (IUMS) under grant number 1402-3-68-26809.
None.
This is an in vitro study. The Iran University of Medical Sciences Research Ethics Committee has approved under IR.IUMS.REC.1399.913 code.
