Pa ge 1 Pa ge 25 American Journal of Life Science and Innovation (AJLSI) Preparation and Characterization of Zinc Oxide Nanoparticles by Co-Precipitation Method and Evaluation of Theirs Antifungal Activity in Spore Germination of Dermatophytes Hussein A. R. Abbood1, Salah S. Zainalabden1 Volume 3 Issue 2, Year 2024 ISSN: 2833-1397 (Online) DOI: https://doi.org/10.54536/ajlsi.v3i2.3183 https://journals.e-palli.com/home/index.php/ajlsi Article Information ABSTRACT Received: June 20, 2024 Accepted: July 24, 2024 Published: July 27, 2024 This research study synthesized Prepared zinc oxide nanoparticles using the co-precipitation method. The antifungal activities of these nanoparticles (ZnO NPs) and their mode of action against Trichophyton rubrum and Microsporum canis were investigated using samples obtained from patients referred to consulting clinics in Kirkuk city. These nano oxides were identified through XRD diffraction analysis, FTIR measurement, and Scanning Electron Microscopy (SEM). The Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values were recorded as 1.330 and 4.530 µg/mL for Trichophyton rubrum, and 0.333 and 10.67 μg/ml for Microsporum canis, respectively. The antifungal effect of (ZnO NPs) was similar to that of Griseofulvin, while it exhibited a higher effect than ketoconazole. Furthermore, (ZnO NPs) demonstrated a significant (P < 0.05) inhibition effect on spore germination for all tested dermatophytes, although the extent of this effect varied depending on the fungal isolates. Keywords Dermatophyte, Spore, Nanoparticle, ZnO, Antifungal INTRODUCTION Dermatophytes, a large group of fungi responsible for causing dermatophytosis or ringworm(Bouchara &Mignon, 2017). This group includes three main genera: Trichophyton, Microsporum, and Epidermophyton spp (Al- Janabi, 2014). Griseofulvin is considered one of the most widely used treatments for dermatophytosis(Gupta et al., 2009) (Barros et al., 2007), and therefore it was very important to increase its effectiveness through the development of nanoparticles(Zili Z et al., 2005). Solid nanoparticles loaded with griseofulvin in the form of a gel showed very, very effective pharmacological capabilities against the pathogenic fungus(Aggarwal &Goind, 2013). Microsporum canis Zinc oxide nanoparticles (Zno-NPs) showed an effective effect against dermatophytes and show stronger inhibition compared to antifungals(Ahmadpour et al.,2021) (El-Diasty et al., 2013) Many antifungals have shown resistance against skin fungi, which is considered a major challenge(Elad et al., 1992) Therefore, it was important to prepare drugs for pathogenic fungi that replace current medications. Therefore, researchers have paid great attention to nanoparticles due to their high effectiveness due to their unique physical and chemical properties(Vatsha B et al., 2013). Many researches have indicated There are many different types of nanomaterials, including silver nanoparticles(Abbood & A. H. S, 2020) and zinc nanoparticles(Liu et al., 2009), which have shown antifungal and antibacterial activity. Zinc oxide nanoparticles (ZnO NPs) are known for their ability to create large surface areas and unique crystalline structures. These metal oxides are highly ionic(Khaleel et al.,1999), unlike organic materials, possess exceptional durability, selectivity, and thermal resistance. Incorporating zinc oxide into daily zinc supplements is common due to its vital role in maintaining human health. Additionally, ZnO NPs have shown compatibility with human cells (Padmavathy &Vijayaraghavan, 2008). The antimicrobial properties of ZnO bulk powder have been extensively studied, demonstrating its effectiveness against bacteria and fungi. In the agricultural industry, zinc compounds are primarily used as fungicides (Bloom & Markson., 2001) . MATERIALS AND METHODS Isolation and Diagnosis of Fungi Samples were obtained from patients who were referred to consulting clinics in the city of Kirkuk. The samples were collected either by scraping from the affected area or by cutting the affected part of the hair and nails. Afterward, the samples were treated with 10% KOH to analyze the keratinous material present. This analysis aimed to identify any highlights and spores. The isolated samples were then transferred to sterile Petri dishes containing Sabouraud’s Dextrose Agar (SDA) medium, supplemented with 50 mg of the antibiotic Chloramphenicol. This was done to facilitate the growth of fungi without any interference from bacteria and yeasts. Subsequently, the isolated samples were sent to the Mycology Laboratory at the Alqalam of the university. They were placed in an incubator set at a temperature of 27-30°C for a duration of two weeks. Following the incubation period, the colonies were carefully tested (Figure 1), and additional tests were conducted to confirm the diagnosis (Table 1) (Khan et al., 2021). 1 Department of Biology, College of Education for Pure Sciences, Kirkuk University, Kirkuk, Iraq * Corresponding author’s e-mail: hussenabdulrazzaq@uokirkuk.edu.iq Pa ge 26 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 3(2) 25-32, 2024 Inoculum Preparation In light of the need to obtain reproducible and comparable results in antifungal susceptibility testing, it is important to standardize the inoculum. Based on the CLSI guidelines, the tested dermatophyte inoculum suspensions were made (Wang et al., 2018). To induce sporulation, colonies grown on PDA for 7-15 days were flooded with 10 ml of distilled water. The use of a sterile loop to mechanically break up the colonies yielded a suspension that contained some tiny fragments of hyphae and conidia. This mixture was then aliquoted into sterile tubes and allowed to settle at room temperature which facilitated sedimenting of larger particles. The suspension was then diluted to a final conidia concentration of 1-3 × 103 cells/ml using a haemocytometer according to CLSI M38-A2. The Dilution and Preparation of Stock Solution of the Antifungal Agents In performing the comparison with (ZnO NPs), commercially available antifungal reference powders of Griseofulvin and ketoconazole were used to prepare stock solutions. Another accurate way of measuring the antifungal powders was by using an analytical balance that measures up to 0. 0001g, according to the authors of the paper by (Wang et al., 2018). Deionized distilled water was employed for dissolving Griseofulvin, ketoconazole, and (ZnO NPs) to prepare stock solutions. To reach the final strength as required, further dilutions were made on the broth medium, to attain twice the strength. Subsequently, a twofold serial dilution was performed to obtain the final concentration range of the drugs: It was found to be 0.04-19 µg/ml for ketoconazole and 0. 136-59 µg/ml for Griseofulvin and 0.171-75 µg/ml for (ZnO NPs). Preparation of Zinc Oxide Nanoparticles by Co- Precipitation Method A solution of 0.4 molar aqueous zinc acetate (Zn(CH3COO)2 2H2O) was created by dissolving 9.2 g of the compound in 100 milliliters of non-ionic water and allowing it to dissolve completely on a magnetic stirrer for 30 minutes. Sodium hydroxide with a concentration of 1 molar was then added drop by drop until reaching a pH of 9. The solution was stirred at 70°C for 2 hours until a precipitate formed. The precipitate was filtered, washed with nonionic water and ethanol, and then dried at 80°C for 30 minutes. Then, the precipitate was burned at 500°C for 5 hours and the process was further continued for desiccation of the precipitate at room temperature for overnight (Khan et al., 2015) . Zn(CH3COO)2·2H2O+ 2NaOH →ZnO + 2CH3COONa + 3H2O Table 1: Shows species and fungal species isolated from spices Mold type Hair penetration test Growth at a temperature of 37°C Diagnosis of large conidia Diagnosis of small conidia Rice test Characteristics of fungal culture M.canis + - Fusiform, thick, rough-walled, with a curved or hooked end It is not found in abundance, and if it is found, it has a club shape Yellow colony tapering at both ends; spindle-shaped T.rubrum + - It takes the form of a cigar or a pen Club-shaped to barrel-shaped, growing along the spindle filaments - The colony gives off a red dye. Below the plate, the conidia are club-shaped and the hyphae are tree-shaped Figure 1: Colony morphology of the A. T.rubrum and B. M.canis isolate Pa ge 27 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 3(2) 25-32, 2024 Nanoparticle Characterization Techniques Based on Zinc Oxide Nanoparticles are Being Prepared Fourier Transform Infrared Spectroscopy (FTIR) Spectroscopy of infrared radiation was used to analyze FTIR samples and the presence of functional groups and various features of the nanocomposite was studied (Fadlelmoula et al., 2022). Scanning Electron Microscope (SEM) Analysis It is applied for the average particle size analysis of the nanocomposite and the structure shape analysis. XRD was also employed to determine what elements are present in the sample. The results were taken and put down in pictures (Xiong et al., 2020) X-Ray Diffraction Analysis (XRD) Analysis based on X-ray diffraction method is very popular technique for testing and determining the crystal structure of nanomaterials by using an XRD tool and it is a non-destructive way to characterize the physical properties, chemical composition and crystal structure, In addition atomic arrangement and thickness could be determine ( Zhang et al., 2023). Determination of MIC Values for Dermatophytes The MIC (ZnO-NPs) and other antifungal drugs considered were assessed against the fungal strain by employing broth microdilution method. The method followed Clinical Laboratory Standards Institute (CLSI) guidelines wherever possible, as described in Filamentous Fungi Document M38-A2 (Badiee et al., 2013). The Minimum Fungicidal Concentration (MFC) When testing MIC, the minimum fungicidal concentration (MFC) of ZnO NPs against the dermatophytes studied was determined. For comparison, the bactericidal efficacy of griseofulvin and ketoconazole was also examined. Samples from wells with no visible growth were then transferred to SDA plates. Additionally, positive controls (from growth control wells) and negative controls (from sterile control wells) were included in the experiment. The Impact of Biosynthesized Zinc Oxide Nanoparticles (Zno Nps) on the Germination of Dermatophyte Spores Was Assessed Using as Microscopic Method To generate the conidia, the isolates were cultured on potato dextrose agar (PDA) for 10-14 days, following the specific requirements of each dermatophyte genus. The resulting suspension contained both conidial and mycelial fragments. The spore count for each dermatophyte isolate was maintained at 40-60 spores per microscopic field. In order to interact with the ZnO NPs, the spore suspension for each isolate was prepared, resulting in a concentration of 20-30 spores per microscopic field. The tubes containing the culture mixture were then placed in a shaking incubator at 35ºC for Trichophyton spp. or at 30ºC for Microsporum spp. to ensure even distribution of the ZnO NPs. The germination of spores was studied through the daily microscope examination of the sample. The spore was deemed germinated once the length of the germ tube reached 1.5 times the diameter of the spore (Plascencia‐ Jatomea et al. ,2003). The antifungal drugs and sterile broth were used as substitutes also to the (ZnO NPs) in the positive and negative controls. All the treatments were performed in three replications. The percentage of spore germination inhibition was determined with the help of the formula: % spore germination inhibition = SC - CT/ SC × 100 *SC: average of spores germinated in control. *CT: average of spores germinated in treatment. RESULTS AND DISCUSSION Diagnosis of Nano-Zinc Oxide by XRD The X-ray diffraction analysis was conducted to characterize the crystal structure and phase purity of the synthesized ZnO nanoparticles. Figure (2) illustrates that the X-ray diffraction spectrum of the zinc oxide sample aligns with the standard spectrum of Zinc oxide as per the database (ZnO JCPDS 36-1451) (Wirunchit et al., 2021). Figure 2: X-ray diffraction (XRD) of silver nanoparticles ( ZnO NPs ) Pa ge 28 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 3(2) 25-32, 2024 FTIR Measurement Results for the Prepared Oxides The infrared spectra of Zn(CH3COO)2.2H2O, which is the aqueous zinc acetate salt, are utilized in the preparation of nano-zinc oxide prior to conducting the reaction. This information is depicted in Figure (3). The results obtained from the experiment lead us to the conclusion that the protein present in the reaction medium serves as a reducing agent and forms a protective layer around the biosynthesized ZnO NPs, known as capping proteins. This capping protein plays a crucial role in preventing the undesirable agglomeration of particles in the medium, thereby ensuring the formation of highly stable ZnO NPs(Hadi&Kadhim, 2019). One significant advantage of using capping protein over polymer and surfactant, which are commonly used as capping agents in the preparation of ZnO NPs, is that it is cost-effective, safe, eco-friendly, and does not require any special conditions. Additionally, capping protein acts as an anchoring layer for the transportation of drugs or genetic materials into human cells (Hu et al., 2011), surpassing the capabilities of surfactant and polymer. Moreover, the presence of a non-toxic protein cap enhances the uptake and retention of these materials inside human cells (Rodríguez et al., 2013). Figure 3: FTIR spectrum of ZnO NPs Figure 4: Scanning electron microscope (SEM) of ZnO NPs Diagnosis of Nano-Zinc Oxide by SEM The scanning electron microscope (SEM) was utilized to observe the morphological and structural properties of nanozinc oxide (ZnO). The nanometer range preparation of nanoparticles is evident in Figure (4). The SEM images revealed that certain nanoparticles were adequately dispersed, while the majority were found in agglomerated form. The agglomeration is attributed to both electrostatic effects and the presence of the aqueous suspension, which demonstrates the behavior of nanoparticle agglomeration. This finding aligns with previous studies on nanoparticle agglomeration (S Zain- Al-Abddeen et al., 2017) (Gaikwad et al., 2015). Determination of MIC and MFC Value Against Dermatophytes The antifungal efficacy of ZnO NPs was studied against Trichophyton rubrum and Microsporum canis. Griseofulvin and ketoconazole, commonly used antifungal medications, were employed as positive controls for comparison. The growth inhibition effect of ZnO NPs on the mentioned fungi was determined through the broth microdilution method to establish the MIC. The MIC values for the tested substances are detailed in Table 2. The findings indicated that ZnO NPs exhibited antifungal properties against all tested dermatophytes within the range of 0.171-75 µg/ml, with varying susceptibility levels based on the fungal species. Griseofulvin, with an MIC range Pa ge 29 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 3(2) 25-32, 2024 of 0.136-59 µg/ml, only demonstrated antifungal activity against M. canis and T. rubrum, with MIC values of approximately 10 µg/ml and 32 µg/ml, respectively. Ketoconazole, having an MIC ranging from 0.04 -19 µg/ ml, exhibited a comparable antifungal effect to that of zinc oxide nanoparticles (ZnO NPs). Statistical analysis indicated no significant variance between the two. The most effective MIC of ketoconazole, at 0.6 µg/ml, was observed against M. canis. The biological activity of zinc oxide is attributed to the disruption of membrane lipids, which led to leakage of cell contents and ultimately cell death (Xie et al.,2011) Zinc oxide can also generate hydrogen peroxide and Zn+2 ions, which cause cell death through a change in cellular metabolism. Preliminary studies indicate that the antimicrobial properties of ZnO NPs are due to the formation of free radicals on the nanoparticle surface. These free radicals are thought to damage lipids in the bacterial cell membrane, causing the membrane to leak and rupture (He et al., 2017) (Reddy et al., 2007). Table 2: Comparative MIC value of biosynthesized ZnO NPs, Griseofulvin and Ketoconazole against T.rubrum and M.canis Tested fungal strains MIC/Mean(µg/ml) ZnO NPs 0.171-75 Griseofulvin (0.136-59) pvalue (ZnO NPs) 0.171-75 Ketoconazole (0.04-19) pvalue T.rubrum 1.330 10 0.0439 1.330 1 >0.05 M.canis 0.333 32 <0.001 0.333 0.6 >0.05 The antifungal properties of ZnO NPs and standard antifungal medications were evaluated concurrently with their minimum inhibitory concentration against the dermatophytes being studied. Figures (5) and (6) show that the MFCs obtained are significantly higher than the MICs. T.rubrum was found to be most susceptible to ZnO NPs, with a minimum fungicidal concentration (MFC) of 4.530 µg/ml, compared to an MFC of 35 µg/ml for Griseofulvin and 4 µg/ml for Ketoconazole. For M.canis, the MFC of ZnO NPs was 10.67 µg/ml, while the MFC of Ketoconazole was 3 µg/ml. Figure 5: Sensitivity profile of T.rubrum to the antifungal agents Figure 6: Sensitivity profile of M.canis to the antifungal agents Pa ge 30 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 3(2) 25-32, 2024 Effect of ZnO NPs on Spore Germination of Dermatophytes The impact of ZnO NPs on the germination of spores from various dermatophytes is outlined in Table (4) The study demonstrated that ZnO NPs exhibited a notable and significant inhibitory effect on spore germination (P < 0.05) in all the dermatophytes tested. The impact of ZnO NPs on fungal strains differed in magnitude, as indicated in Table 4. Additionally, the majority of germinated spores exhibited morphological transformations, including spore enlargement. These alterations align closely with the fungistatic properties of ZnO NPs. Consequently, it is postulated that the mechanism behind the fungistatic effect involves modifications in the osmotic balance and permeability of the spores (Khalil et al., 2013). Table 3: Comparative MFC value of ZnO NPs, Griseofulvin and Ketoconazole against T.rubrum and M.canis Tested fungal strains MIC/Mean(µg/ml) ZnO NPs 0.171-75 Griseofulvin (0.136-59) pvalue (ZnO NPs) 0.171-75 Ketoconazole (0.04-19) pvalue T.rubrum 4.530 35 <0.001 4.530 4 >0.05 M.canis 10.67 >64 <0.001 10.67 3 >0.05 Table 4: Effects of ZnO NPs on spore germination in the tested dermatophytes, alongside the reference antifungal drugs Griseofulvin and Ketoconazole Tested fungal strains Mean percentage of spore germination inhibition ZnO NPs Griseofulvin pvalue (ZnO NPs) Ketoconazole pvalue T.rubrum 98 0 <0.0001 98 0 <0.0001 M.canis 88 0 <0.0001 88 0 <0.0001 CONCLUSION In the results obtained in this study and through the effectiveness of ZnO NPs on two types of fungi and comparing them with antifungals, we can conclude that ZnO NPs have a biological effectiveness more or similar to antifungals. 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