Comparison of antifungal and cytotoxicity activities of titanium dioxide and zinc oxide nanoparticles with amphotericin B against different Candida species: In vitro evaluation

Abstract Background Candida species are known to cause serious fungal infections that produce cutaneous, mucosal, and systemic infections. Nowadays, mortality and morbidity candidiasis in immunocompromised patients have increased. Nanotechnology is a new world‐known technology and includes particles ranging from about 1 to 100 nanometers. The purpose of this study was to evaluate the antifungal and cytotoxicity activities of titanium dioxide nanoparticles (TiO2‐NPs) and zinc oxide nanoparticles (ZnO‐NPs) compared to amphotericin B (AmB) on different Candida spp in in vitro conditions. Methods In the present study, susceptibility of different Candida species to TiO2‐NPs and ZnO‐NPs compared to AmB was determined by broth microdilution (BMD) and agar well diffusion methods. Cytotoxicity of TiO2‐NPs and ZnO‐NPs and amphotericin B was measured by MTT (3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐Diphenyltetrazolium Bromide) assay. Results The results indicated that the TiO2‐NPs and ZnO‐NPs showed antifungal activities against pathogenic Candida spp. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TiO2‐NP ranges against Candida spp. were 128‐256 µg/mL and 256‐512 µg/mL, respectively. The MIC and MFC values of ZnO‐NPs were 64‐128 µg/mL and 256‐512 µg/mL, respectively. However, MICs and MFCs of AmB were 8‐16 µg/mL and 16‐32 µg/mL, respectively. The MTT assay results showed that the CC50% belonged to ZnO‐NPs 706.2 μg/mL, for TiO2‐NPs 862.1 μg/mL, and for AmB 70.19 μg/mL, respectively. Conclusion Our findings showed that TiO2‐NPs and ZnO‐NPs had antifungal effects against all Candida species, yet the antifungal properties of TiO2‐NPs and ZnO‐NPs were significantly less than those of AmB. The CC50% of AmB was significantly lower than ZnO‐NPs and TiO2‐NPs.


| INTRODUC TI ON
Candidiasis is an opportunistic fungal disease with a great variety of clinical symptoms that can affect various body parts including the skin, nails, oral mucosa, vagina, and internal organs. 1 These infections can be fatal depending on the host's immune system. This infection is known as one of the major important causes of death, particularly among immunocompromised hosts. 2 Predisposing factors of Candida species include avitaminosis, obesity, pregnancy, alcohol consumption, broad-spectrum antibiotics and corticosteroids, physiological changes, and age. 3 Several species of Candida such as C albicans and non-albicans Candida (NAC) species including C tropicalis, C kefyr, C krusei, C glabrata, and C parapsilosis are known as the common cause of candidiasis. 4 Different species of Candida are able to convert to invasive pathogens when the immune system is weakened or the microbial balance of the normal flora of the body is disturbed. 5,6 Topical antifungal drugs such as nystatin, clotrimazole, and miconazole are used to treat superficial candidiasis. For systemic or disseminated Candidiasis, oral ketoconazole, oral and intravenous fluconazole (FLC), and intravenous amphotericin B (AmB) are recommended. 7,8 A major concern of the increasing consumption of chemical drugs like antifungal agents is their side effects, which might in some cases be even more dangerous than the disease itself. Headache, nausea, vomiting, hepatotoxicity, and anaphylaxis are some side effects of these drugs. 9 Other side effects of antifungal drugs include the development of resistant fungal species and therapeutic failures. 1 For these reasons, many studies have been conducted to find new compounds with antifungal effects. 10 They are one of the compounds of nanoparticles. The nanoparticles have been studied both individually and in combination with antifungal drugs to achieve better methods for treating various diseases. Furthermore, the nanoparticles are easily available, more affordable, and more effective with similar effects, which makes them a good alternative to chemical drugs. 11 Titanium dioxide (TiO2) is a nanoparticle, which has been investigated more than any other substance due to its very high photocatalytic activity. 11 It exists in three crystalline phases including anatase, rutile, and brookite the most stable of which, at normal pressure and temperature, is the anatase structure and the other two phases are semi-stable. 12 Some properties of this material that make it preferable to other particles include its high chemical resistance, non-toxicity, endurance, availability, and low production cost. 13 In recent decades, the using of zinc oxide (ZnO-NPs) has increased due to large energy band gaps, chemical-thermal stability, high oxidation dependence (60 mV), and non-toxicity. 14 MTT assay is used to determine the rate of cell proliferation and cell viability, and its mechanism depends on the decrease of insoluble crystals of MTT formazan by the enzyme succinate dehydrogenase in the mitochondria of the cell. Dimethyl sulfoxide (DMSO) solution was used to dissolve these crystals. Then, the amount of light absorption was measured in terms of the intensity of the blue color of formazan at a wavelength of 540 nm. If the cell is alive and reproducing, the rate of dye production and the amount of absorption read are higher, while if more cells are dead and inactive, the rate of light absorption is lower. 15 The aim of this study was to evaluate the antifungal and cytotoxicity activities of titanium dioxide nanoparticles and zinc oxide nanoparticles compared to amphotericin B.

| Preparation and determination of Characterization of TiO2-NPs and ZnO-NPs
TiO2-NP and ZnO-NP powders were purchased from Iranian Nanomaterial Pioneer Company (Mashhad, Iran). Characterization of TiO2-NPs and ZnO-NPs was determined by scanning electron microscope (SEM), UV spectroscopy, and X-ray diffraction (XRD). The SEM micrographs demonstrated the shapes and sizes of the TiO2-NPs and ZnO-NPs. The crystalline structure of these nanoparticles was measured using X-ray diffraction (XRD) (Panalytical, Almelo, Netherlands).

| Candida spp. and growth condition
This study was carried out on five different Candida species isolated from patients with different types of candidiasis. These species were previously identified by real-time PCR High Resolution Melting Analysis and sequencing methods. 6 The species included C tropicalis, C parapsilosis, C krusei, C albicans, and C lusitaniae. First, the Candida spp was subcultured onto Sabouraud dextrose agar (SDA) (Liofilchem Company, Italy).

| Broth microdilution (BMD) method
To determine MIC and MFC values of TiO2-NPs and ZnO-NPs compared to AmB on five different Candida species, the guideline of Clinical and Laboratory Standard Institute (CLSI, M27-ED4 document) was followed. 16  All experiments were carried out in triplicate.

| Measuring cytotoxicity of TiO2-NPs and ZnO-NPs compared to AmB by MTT assay
MTT assay was used to determine cell proliferation and viability of macrophage J/774. 15

| Characterizations of TiO2-NPs and ZnO-NPs
As shown in Figure 1  As shown in Figures 5 and 6, the lowest MIC of TiO2-NPs in C albicans, C krusei, and C parapsilosis was 128 µg/mL and the highest MIC of TiO2-NPs in C lusitaniae and C tropicalis was 256 µg/mL.

Moreover, the results indicated that the highest MFC of TiO2-NPs
for C tropicalis was 512 µg/mL and the lowest MFC of TiO2-NPs in C parapsilosis, C lusitaniae, C krusei, and C albicans was 256 µg/mL.

The minimum amount of ZnO-NPs required for the growth inhibition
of C parapsilosis and C krusei was 64 µg/mL and for C lusitaniae, C albicans, and C tropicalis was 128 µg/mL. The lowest MIC of ZnO-NPs was observed for C parapsilosis and C krusei, and the highest MIC values were obtained for C lusitaniae, C albicans, and C tropicalis.
The lowest MIC value of AmB in C lusitaniae, C albicans, and C tropicalis was 8 µg/mL, and the highest MIC value of AmB in C parapsilosis, and C krusei was 16 µg/mL. The results revealed that the highest MFC of AmB in C tropicalis and C albicans was 32 µg/ mL and the lowest MFC of AmB in C parapsilosis, C lusitaniae, and C krusei was 16 µg/mL.
In the present study, using the agar well diffusion method, the diameters of zones of inhibition of five different candida spp in con-

| MTT assay results
Survival rate of mouse macrophages J/774 exposed to TiO 2 -NPs and ZnO-NPs compared to AmB is shown in Figure 7. The MTT results showed that the CC50% were for ZnO-NPs 706.2 μg/mL, for TiO2-NPs 862.1 μg/mL, and for AmB 70.19 μg/mL, respectively. Our finding indicated that the rate of cytotoxicity increased with increasing concentration. The CC50% of AmB was significantly lower than ZnO-NPs and TiO2-NPs (P < .05). The cytotoxicity ratio ranking on mouse macrophages J/774 was TiO2-NPs > ZnO-NPs > AmB.

| D ISCUSS I ON
In the recent decades, the prevalence of opportunistic diseases such as fungal infections in immunocompromised patients has been increased. Candidiasis is the major common invasive fungal infection in human. 8 Due to the side effects of antifungal drugs and the F I G U R E 3 X-ray diffraction pattern obtained from TiO 2 -NPs F I G U R E 4 X-ray diffraction pattern obtained from ZnO-NPs development of resistant fungal species and therapeutic failures, attention has been drawn to the use of novel antifungal compounds with fewer side effects. 18 One of these novel compounds is na- F I G U R E 7 Survival rate of mouse macrophages J/774 exposed to TiO 2 -NPs and ZnO-NPs compared to amphotericin B and synthesis mode of the nanoparticles as well as the studied Candida species.

| CON CLUS ION
Our finding showed that the TiO 2 -NPs and ZnO-NPs had antifungal potentials against pathogenic Candida spp. and could inhibit the growth of all tested Candida spp. However, its antifungal properties were significantly less than those of AmB. MTT assay results revealed that the rate of cytotoxicity increased with increasing concentrations. Finally, the CC50% of AmB was significantly lower than ZnO-NPs and TiO2-NPs.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R S CO NTR I B UTI O N S
SS and PGHA developed the study concept and design. SHA collected the data. SS analyzed and interpreted the data. SS wrote the article. PGHA and SS revised and edited the article. All authors read and approved the final article.

E TH I C A L A PPROVA L
The study was evaluated and approved by the Ethics Committee of