Antifungal effects of ZnO, TiO2 and ZnO-TiO2 nanocomposite on Aspergillus avus

This study aimed to synthesis ZnO, TiO 2 and ZnO–TiO 2 (ratio weight of 1/1 for Zn/Ti) nanoparticles using zinc acetate and titanium isopropoxide through the sol-gel method. Physicochemical and morphological characterization and antifungal properties evaluation like minimum inhibition concentration (MIC) and minimum fungicide concentration (MFC) of nanopowders were investigated against Aspergillus avus at in vitro. All synthesized nanoparticles (50 µg/ml) showed fungal growth inhibition while ZnO-TiO 2 showed higher antifungal activity against A. avus than pure TiO 2 and ZnO. TiO 2 and ZnO-TiO 2 (300 µg/ml) inhibited 100% of spur production. Pure ZnO and TiO 2 showed pyramidal and spherical shapes, respectively whereas ZnO-TiO 2 nanopowders illustrated both spherical and pyramidal shapes with grown particles on the surface. Based on our ndings, low concentration (150 µg/ml) of ZnO-TiO 2 showed higher ROS production and stress oxidative induction thus fungicide effect as compared to alone TiO 2 and ZnO. In conclusion, ZnO-TiO 2 nanostructure can be utilized as an effective antifungal compound but more studies need to be performed to understand the antifungal mechanism of the nanoparticles rather than ROS inducing apoptosis.


Introduction
ZnO and TiO 2 have been used in various biomedical applications due to photocatalytic, antimicrobial and antifungal properties [1,2]. Doping and nanocomposite manufacturing have been previously utilized as the processes for enhancing the antifungal activity of this kind of nanoparticles. Among the various semiconductor nanomaterials, titanium dioxide (TiO 2 ) and zinc oxide (ZnO) have achieved more attention due to their high chemical stability, nontoxicity, relatively low cost and high antimicrobial activity [3,4].
The metal oxide NPs antibacterial and antifungal properties have been previously studied [5][6][7] and the ndings showed the ZnO antibacterial activity as well its capability to increase of induction of reactive oxygen species (ROS) production by decreasing its particle size. The zinc oxide antifungal activity is related to the formation of free radicals on the surface of nanoparticles that damage the fungal cell membrane lipids, which lead to protein leakage through the membrane disruption [8][9][10][11][12].
TiO 2 NPs possess the antimicrobial properties even at low concentrations through the photocatalytic process that causes fatal damage in treated microorganisms [13][14][15]. Based on TiO 2 nanoparticle antimicrobial properties, these nanoparticles in the anatase and rutile phase show the excellent antifungal properties [16]. The titania owned enormous applications because of its high thermal/chemical stability, and high photocatalytic activity. The toxicity of titania nanoparticles originates from its physical properties, not its chemical structure. These nanoparticles can permeate from biological barriers that can damage the cells or even organs. Some methodologies have been previously applied for improving the titania NPs' antimicrobial activities on simple microorganisms such as bacteria and viruses [17][18][19][20][21].
By considering the Aspergillus species as the deadliest opportunistic fungal infections, these fungi are the main threat to human health. Among the 600 species of Aspergillus, the avus, fumigatus, and niger species possess the pathogenicity for humans and growing on crops can cause the occurrence of some disease [22,23]. Upon the previous quantitative reports on ZnO and TiO2 fungal growth inhibition, these nanoparticles possess fungicidal effects on Candida albicans, Aspergillus niger, and Penicillium sp. fungus. We showed previously the increase of ZnO and TiO 2 antibacterial activity by increasing the concentration of dopant in doped ZnO and TiO 2 [24,25]. This study aimed to synthesize the ZnO, TiO 2 , and ZnO-TiO 2 nanostructures using the sol-gel methodology, physicochemical characterization of nanopowders, and antifungal assays against Aspergillus avus to nd the highly effective antifungal concentration at dark condition.

Nanostructures synthesis
ZnO and TiO 2 nanostructures were synthesized using the sol-gel method as described by Najibi [25]. For the preparation of ZnO-TiO 2 nanostructures, separately prepared ZnO and TiO 2 sols were mixed at the same molar ratio of Zn:Ti then the mixture was stirred at ambient temperature for 2 h and the stirred solution was remained for 24 h to obtain a gel. Prepared gel was dried at 100 °C and was calcined at 500 °C for 2.5 hours.

Material characterization method
The XRD pattern and phase identi cation of nanopowders were determined by X-RAY diffraction analysis (Philips-MPD XPERT, λ: CuKα=0.154 nm) and 20-70˚ range of scanned samples were considered as 2Ө.
The scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analyzer (N5, Backman, USA), and zeta potential analyzer (Malvern Zeta-sizer 3000, Malvern Instrument Inc., London, UK) were utilized for morphological, size, and zeta potential characterization of all samples, respectively. Fourier Transform Infrared (FTIR) Spectroscopy was used to identify organic, polymeric, and in some cases, inorganic materials. Fourier transforms infrared (FTIR) spectra were obtained using a Bruker IFS 48 instrument (Bruker Optik GmbH, Germany). All spectra were taken under air as a function of time with 16 scans at a resolution of 4 cm − 1 and a spectral range of 4000-5000 cm − 1 .

Antifungal assay
A. avus, purchased from the Iranian biological resource center (IBRC), were cultured on Sabouraud dextrose agar (SDA; Merck, Darmstadt, Germany) at 25 °C and the dark condition. The autoclaved SDA media containing ZnO, TiO 2 and ZnO-TiO 2 NPs at concentrations of 0, 37, 75,150 and 300 µg ml − 1 and an NP-free solution were poured onto the 6 cm diameter Petri dishes. To determination of minimum inhibition concentration (MIC) of nanoparticles for each treatment group, the CLSI-M38 standard method was used for the time intervals of 7 days by measuring the diameter of fungal colonies opacity. To determine the minimum fungicide concentration (MFC), the higher concentrations than MIC for each nanostructure were used on SDA medium similar to the MIC determination experiment and the minimum concentration that killed A. avus considered as MFC. To detect the production of ROS after each time point of treatment, 2′-7′-Dichlorodihydro uorescein diacetate (DCFH-DA) solubilized in ethanol (5 µM nal concentration) was added to the cultures and incubated on a shaker at room temperature at the dark condition for 1 h. DCFH-DA, a nonpolar dye, is converted to the non uorescent polar derivative DCFH by cellular esterases. DCFH can switch to highly uorescent DCF through oxidization by intracellular ROS and possessing an excitation wavelength of 485 nm and an emission band between 500 and 600 nm.
After incubation time, samples were subjected to uorescence microscopy (Biozero BZ-8000; Keyence, Osaka, Japan) equipped with the following lter set EX 495 nm EM 510 nm, and uorescence spectrophotometric (RF-5000, Shimadzu, Kyoto, Japan) analysis at room temperature. The XRD patterns of ZnO and TiO 2 showed a single high-intensity peak that implies a highly oriented and single-crystalline nature of the samples. As shown in Fig. 1, the intensity of TiO 2 peaks considerably decreased after the addition of TiO 2 into the structure of ZnO in the ZnO-TiO 2 composite that indicates the greater crystallinity of pure TiO 2 NPs compared to ZnO-TiO 2 NPs [27]. Pro le broadening also indicated the small crystalline domain sizes of wurtzite and anatase indicating that the ZnO-TiO 2 composite hinders the growth of particles during calcination. The main peaks of each sample in the range of 2θ = 20-50° speci ed some peaks belonging to anatase (Fig. 1). Table 1

PSA and Zeta potential analysis
The zeta potential is an important indicator of the stability of dispersed particles in the suspension solution. The zeta potential determines the repulsion of dispersed particles in the solution. Small particles require the high zeta potential for superior stability, and low zeta potential causes to particle accumulation. The zeta potential of a particle alters by the particle surface chemical composition, the pH and ionic strength of the solution. Zeta potential of ZnO, TiO 2 , and ZnO-TiO 2 were − 11.6, -36.4, and − 12 mV, respectively ( Fig. 2 and Table 1). Based on our ndings, TiO 2 and ZnO-TiO 2 showed the highest and lowest stability in aqueous suspension, respectively. Larger particle sizes for ZnO (608 nm), TiO2 (299 nm), and ZnO-TiO2 (983 nm) were determined by PSA analysis showing the agglomeration of nanoparticles.

SEM and TEM analysis
As shown in Fig. 3, the ZnO and TiO 2 nanoparticles illustrated hexagonal-pyramidal and spherical shape with grown articles on surfaces, respectively. The wurtzite-structured ZnOcrystal is described as several alternating planes composed of four-fold tetrahedrally-coordinated O 2− and Zn 2+ ions stacked alternatively along the c-axis [28]. The oppositely-charged ions produce positively-charged Zn (0001) and negatively-charged O(0001 ) surfaces, resulting in a normal dipole moment and spontaneous polarization along the c-axis, as well as a divergence.
In the ZnO-TiO 2 nanostructures, the morphology was a mixture of pyramidal and spherical with more agglomeration while the particle sizes were smaller than alone titanium and zinc oxide particles. Upon the EDX analysis, the strong signals of Zn, Ti and Zn-Ti were observed in ZnO, TiO 2 , and ZnO-TiO 2 nanostructures, respectively (Fig. 3).
The TEM images of nanostructures clari ed the regular growth of all nanostructures and illuminated the TiO 2 (5 nm) particle size smaller than ZnO (10 nm) and ZnO-TiO 2 (35 nm) nanoparticles with lower agglomeration rate (Fig. 3). related to the hydroxyl groups. Also, water molecules in the bending band at 1630 cm − 1 are visible [31]. The presence of some bands can be associated with the organic phase of solid, despite the use of organic compounds in the synthesis of nanoparticles (Fig. 4).

Antifungal properties of nanostructures
As shown in Table 1, ZnO-TiO 2 nanostructure exhibits better antifungal effects against A. avus than other nanoparticles due to its high speci c surface area. By increasing the speci c surface area, the possibility of chemical reactions and the production of reactive oxygen species on the surface were increased [32]. The MIC for ZnO-TiO 2 , ZnO, and TiO 2 against A. avus was determined 39, 156, and 78 µg/ml, respectively. Because of the small particle size, the best cell internalization, and the ability to produce more reactive oxygen species, TiO 2 showed a higher fungicide than ZnO. The MFC for ZnO, TiO 2 , and ZnO-TiO 2 was 312, 156 and 78 µg/ml, respectively. The particle size of the ZnO-TiO 2 nanostructure possessed a sharp structure with smaller particles than the cell membrane that can inhibit the growth of the fungus by entering the cell membrane and injuring the cell wall thus resulting in the high toxicity. Figure 5 illustrated the inhibition zone of ZnO, TiO 2 , and ZnO-TiO 2 at 37.5, 75, 150, and 300 µg/ml concentrations. By increasing the concentration of nanoparticles, inhibition zone diameter of growth increased and 100% of inhibition was achieved at 300 µg/ml for TiO 2 and ZnO-TiO 2 treated groups. The minimum fungal growth (72%) was obtained at 37.5 µg/ml for ZnO-TiO 2 while for ZnO was 50% at the same concentration showing that the TiO 2 synergistic effect into the mixture [33]. Among all nanoparticles, ZnO nanoparticles showed the lowest fungicide activity compared to others whereas it signi cantly increased the antifungal activity in ZnO-TiO 2 nanocomposite.
The destructive changes were observed on the shape and growth of the treated A. avus (at a concentration of 37.5 µg/ml for all samples) compared to the untreated control group. As shown in Fig. 6, the untreated control fungus produced the highest count of fungal spores while treated groups showed a lower count of spores and damaged tubular laments, in instance deformation, smoothness, and noticeably thinner in hyphae compared to the untreated group. Upon the previous reports, increasing the hyphae causes to form whiter medium [34] and our ndings agreed to color changes based on the used nanoparticles (Fig. 6).
Among the reactive oxygen species, hydrogen peroxide and hydroxyl radicals as the strong and nonselective ROSs can damage all types of biomolecules including carbohydrates, acids, lipids, proteins, DNA, RNA, and amino acids through inducing the oxidative stress [35]. The production rate of the three  [36]. There is a direct dependency between increasing the formation of ROS and the fungicide of nanoparticles. As shown in Fig. 7, all nanoparticles raised the ROS production in treated A. avus compared to untreated control with order ZnO-TiO 2 > TiO 2 > ZnO > untreated control. The production of intracellular ROS was in uenced by the type and speci c surface of nanoparticles. Titania can produce ROS higher than zinc oxide [37], our ndings also con rmed the highest ROS production through stronger uorescence intensity in ZnO-TiO 2 treated group. In ZnO-TiO 2 nanostructures, the speci c surface area is higher than other nanoparticles (TiO 2 and ZnO) and accordingly high ROS generation. Oxidative stress induced by reactive oxygen species generation in ZnO-TiO 2 nanostructures is thought to be the main mechanism of antifungal activity. The suggested mechanism for the antifungal activity of these compounds can be based on the formation of high levels of reactive oxygen species (ROS) that disrupt the integrity of the fungal cell membrane, which assists in the damage of microbial enzyme bodies thus killing the fungi [38].

Conclusion
This study aimed to compare the antifungal properties of TiO 2 and ZnO versus ZnO-TiO 2 nanocomposites to select the compound with the highest antifungal activity. Based on our ndings, low concentration (150 µg/ml) of ZnO-TiO 2 showed higher fungicide and stress oxidative induction through ROS production as compared to TiO 2 and ZnO. In conclusion, ZnO-TiO 2 nanostructure composition can be used as an effective antifungal compound but more studies need to be performed to deeply understand the antifungal mechanism of the nanoparticles rather than stress oxidative induction.

Declarations
Contributions NNI carried out most of the experiments and wrote this paper. MM participated in this project and proposed the idea. AYKH designed and supported the project edited and revised the paper. All authors read and approved the nal manuscript.

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