Bioengineered synthesis of iron oxide nanoparticles using Morchella conica: Characterization and evaluation of biomedical applications

2.1 Morchella conica collection and extract preparation

Morchella conica (Morchellaceae) was collected from District Bajaur, Tehsil Salarzai village, Batwar in the spring season (2022-2023). Bajaur District, located in Pakistan’s North West between 34-72” and 34-74” N and 71-50” and 71-54” E longitudes, is the entrance point to all of Ex-Fata and Khyber Pakhtunkhwa (Khan, 2022). The collected and identified mushroom species were properly cleaned with tap water to obtain clear of any surface contaminants. Morchella conica was shade-dried at room temperature for about 3-4 weeks and ground into fine powder (Hameed et al., 2019; Abdelshafy et al., 2022). For the preparation of Morchella conica extract, 30 g powder was carefully added into a 500 mL flask containing 400 mL of distal water along with a magnet and placed on a hotplate at 80°C for 2 h. The extract was cooled and filtered three times using Whatman filter paper to obtain a pure aqueous extract (Iqbal et al., 2020). After that, pH was recorded at room temperature, and finally, the mushroom extract was stored at 4°C for further studies.

2.2 Synthesis of IONPs

To achieve the purpose of iron NPs from the prepared Morchella conica extract, the acetate precursor salt was utilized. The help of biological synthesis of nanoparticles was done by reducing iron salt. 3 grams of iron salt were steadily mixed with 250 mL filtered Morchella conica extract. To create a standardized solution, the solution mixture was continuously heated for 2 h at 80°C. After that, it was centrifuged for 20 min at 6000 rpm. Following centrifugation, the supernatant was disposed of, and the pellet, which was thought to be IONPs, was carefully cleaned three times with distilled water to get rid of any last contaminants. After that, isolated powder NPs were incubated in a hot air incubator at ∼100°C until the water evaporated completely. The synthesized IONPs were annealed at 400°C to obtain pure crystalline NPs. IONPs were then comprehensively characterized utilizing a variety of characterization methods after the collected powder samples were stored in dry, cold, and dark conditions in airtight vials (15 mL falcon tubes) (Iqbal et al., 2019; Abbasi et al., 2023).

2.3 Characterization of IONPs

The biogenic IONPs were characterized using different characterization techniques. The absorption spectra were measured for determining the bio-reduction of iron ions to IONPs via a UV-4000 UV-Vis spectrophotometer (Lambda 35^® PerkinElmer, USA) within the ranges of 200-700 nm (Chatterjee et al., 2020). The elemental composition and morphological study of IONPs were done using SEM (NOVA FEISHM-450), and the FT-IR (Alpha, Bruker, Germany) investigation was achieved from 1000-4000 cm^-1, finding out diverse functional groups that control the reduction and effective stabilization of IONPs. The crystal structure of the biogenic IONPs was found out by using analytical XRD (Netherlands), and after that, the crystal size was deliberate by the help of XRD (Abbasi et al., 2019).

2.4 Antibacterial activity

Using the disc diffusion method, the antibacterial activity of IONPs was evaluated against three bacterial strains: Klebsiella pneumoniae (ATCC 4617), Bacillus subtilis (ATCC 6633), and Escherichia coli (ATCC 15224). Nutrient agar plates were equally covered with a 0.1 mL bacterial suspension that had been standardized to 1.0 McFarland turbidity. In accordance with the method outlined by Ansari et al. (2017), sterile 6 mm discs were impregnated with varying concentrations of IONPs (ranging from 47 μg/mL to 1500 μg/mL) and subsequently placed on the inoculated agar surfaces. To assess the antibacterial activity of the produced IONPs, the plates were incubated for 24 h at 37°C. After this, the inhibitory potential zones were assessed in millimeters. The assessments were carried out in a triplicate manner. Amp-B (10μg) was taken as a positive control, and maximum inhibition concentration (MIC) standards of Morchella conica-mediated IONPs were calculated by calculating the zone of inhibition (ZI) (Dharmaratne et al., 2018; Iqbal et al., 2019).

2.5 Antioxidant activity of IONPs

The antioxidant activity of the synthesized IONPs was evaluated by means of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging examination, following the procedure drawn by Yap et al. (2021). Numerous concentrations of IONPs (ranging from 4 to 500 μg/mL) were developed by using distilled water. For each test, 1 mL of the IONP suspension was mixed with 1 mL of fresh prepared DPPH (0.1 mM) solution. The mixtures were vortexed slightly and put to incubate in the dark for 32 min to stop photodegradation. Using Shimadzu UV-Visible spectrophotometer, absorbance readings were recorded at 517 nm. Accuracy was ensured by performing experiments in triplicate. A solution comprising only DPPH was used as the negative control sample, while ascorbic acid was employed as the positive reference standard material. The percentage (%) of DPPH radical scavenging was calculated accordingly (Sridhar and Charles, 2019; Ray et al., 2024).

$\text{Absorbance-Absorbance=}\frac{\left[\text{Sample/standard}\right]\text{\ }}{\text{[Absorbance\ of\ control]}}\times \text{\ 100}$

2.6 Anticancer activity

To evaluate the anticancer potential of synthesized IONPs, experiments were conducted using the HepG2 human liver cancer cell line. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to ensure optimal growth conditions. Once a sufficient number of cells were cultured, they were seeded into 96-well microplates and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ to promote cell attachment. Following the incubation, the cells were exposed to IONPs across a concentration gradient ranging from 3.91 to 500 µg/mL. Cisplatin was used as a reference drug (positive control) for comparative purposes. After treatment, 100 µL of MTT reagent was added to each well, and the plates were incubated for an additional 2 h to allow the formation of formazan crystals by metabolically active cells. Post-incubation, the supernatant was carefully discarded, and 100 µL of DMSO was introduced into each well to dissolve the formed formazan. The optical density, indicative of cell viability was measured at a wavelength of 570 nm using a microplate reader. Finally, the half-maximal inhibitory concentration (IC₅₀) of the IONPs against HepG2 cells was determined based on the absorbance data (Gawel et al., 2022; Zhi et al., 2020).

2.7 Antifungal activity

To find out the antifungal activity of IONPs, the concentration range from 47 up to 1500 µg/mL was selected for the fungal pathogens test. In this study involved three different fungal strains. Aspergillus flavus, Mucor racemosus, (FCBP: 0300) and candida albicans, (FCBP: 478) these three strain were selected to provided a broad representation of pathogenic fungi. The investigational design aimed to find out the fungicidal potential of IONPs by studying their inhibitory effects across diverse dosages. Sabouraud dextrose agar (SDA) was prepared and autoclaved following the manufacture pattern (Ray et al., 2024). Sterile Petri plates were filled with medium (15-20 mL) and left to harden for 15 to 20 min.

Then, 100 mL cotton swabs were used to create identical lawns in Petri dishes using diverse pathogenic fungal strains. Resting on Petri dishes, filter discs with different concentrations of Morchella conica asynthesized IONPs were maintained. Antifungal drug (Amp B) was used as a control. The Petri plates were placed in a 37°C heated incubator for 24 h. The ZIs were measured in millimeters, and the minimum inhibitory concentration (MIC) value was calculated. (Ahmad et al., 2022).

2.8 Cytotoxicity assay against brine shrimps

To study the cytotoxic potential of fabricate IONPs the BSLA was used. The eggs of Artemia salina (Ocean Star, USA) was used for this purpose and was incubated for 24 to 48 H at 30°C under light in seawater (3.8 g/L) for this activity. Using a Pasteur pipette, 10 mature grown phototropic Nauplii were harvested and placed into glass vials filled with IONPs and artificial seawater (Hnamte et al., 2020). IONPs were prepared at various concentrations (ranging 4 to 500 µg/mL) for cytotoxic evaluation. For the positive control, vials include vincristine sulfate, saline solution, and Artemia nauplii (brine shrimp larvae). After a 24-h exposure period, the mortality among the nauplii was recorded in all treatment groups’. The quantity of deceased larvae in each vial was counted, and the data were analyzed using GraphPad software to calculate the median lethal concentration (IC₅₀), as described by Iqbal et al. (2020).

2.9 Statistical analysis

ANOVA was used to compare the groups statistically, and Tukey’s post hoc test was used to find significant differences. A p-value of less than 0.05 was deemed statistically significant.

3.1 UV-spectrophotometery

A UV-spectrophotometer was used to confirm the fabrication of IONPs. At a specified wavelength range of 200-800nm, the spectrum was measured. At 302 nm, the broad peak represents maximum absorption. Because of the surface Plasmon resonance (SPR) excitation that caused the color changes and IONP fabrication, the peak was realistic. The range of peaks for IONPs was also reported by Yap et al., 2021. Fig. 1 shows the UV-spectrophotometry for the Morchella conica mediated IONPs. The development of peak is dictated by different factors like shape, size, and polydispersity of IONPs.

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Fig. 1.

UV-Vis spectroscopy for IONPs from Morchella conica.

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3.2 SEM analysis

SEM was used to analyse the surface morphology of the synthesized IONPs, as shown in Fig. 2. The majority of the NPs were spherical, and there was obvious clumping. Electrostatic interactions between the surface layers of the NPs were probably the cause of this clustering behavior. Furthermore, the nhigh surface area-to-volume ratio tends to encourage aggregation in suspension (Jan et al., 2021).

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Fig. 2.

The SEM analysis of Morchella conica-mediated IONPs.

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3.3 FTIR analysis

FTIR analysis was used to identify the different functional groups responsible for the synthesis of IONPs, as shown in Fig. 3. The IONPs displayed absorption peaks at 357.93, 3290.21, 2842.12, 2202.68, 1726.28, 1022.11, 658.33, and 549.65 cm¹, each corresponding to different functional groups. Furthermore, due to their tiny size, NPs have a high surface area-to-volume ratio, which enables them to interact with biological molecules to produce the desired effects. Size and shape play a critical role in defining their functions in biological applications (Hameed et al., 2019).

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Fig. 3.

FT-IR analysis using for functional groups of Morchella conicaIONPs.

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3.4 X-ray diffraction analysis

The crystal structure of the biogenic IONPs was determined by using analytical XRD (Netherlands), and after that, the crystal size was calculated (Abbas et al.,2019). The XRD pattern of bio-fabricated IONPs reveals orientation planes at (012), (110), (113), (024), (018), and (214) corresponding to 2θ values of 22.03°, 36.93°, 41.40°, 44.1°, 57.9°, and 62.89°, respectively (Fig. 4). The average size was calculated as 19.78 nm. The absence of Bragg peaks for other related compounds indicates that biogenic IONPs are pure-crystalline. The XRD pattern for IONPs is related to previous results of (Jan et al., 2021; Kapoor et al., 2023).

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Fig. 4.

XRD analysis confirms the crystal size of IONPs.

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3.5 Anti-bacterial assay

The antibacterial potential of Morchella conica-mediated IONPs has been tested against numerous bacterial strains, including Escherichia coli, Bacillus subtilis, and Klebsiella. Pneumoniae, with concentration ranging from 47 to 1500 μg/mL, by the disc diffusion method, as shown in Fig. 5 and Table 1. The ZIs were measured, and the MIC values were recorded. The most sensitive strain was Bacillus subtilis (47 ±3.1 μg/mL). Escherichia coli and Klebisella pneumoniae were found to be at 94 ± 2.7 μg/mL, and 188 ± 5.2 μg/mL, respectively (n = 3, p < 0.05). One-way ANOVA showed significant differences in MIC values between bacterial and fungal strains. For the positive control, oxytetracycline (10 µg) was used. As compared to the positive control, none of the concentrations showed more potential. The synthesized IONPs have shown potential for antibacterial activity against various bacterial strains (Jan et al., 2021). Different functional groups present on the surface of NPs account for the enhanced antibacterial capability of IONPs. To put it for a short time, IONPs concluded that their response to the various fungal strains was concentration-dependent (Kamal et al., 2023). As the concentration increased, the antibacterial potential also gradually increased. Other researchers have studied the anti-bacterial properties of IONPs, and established that the primary mechanism supplying NPs with anti-bacterial potential and inducing cell death is ROS production (Nayak et al., 2024). Moreover, studies have shown that NPs give the impression of staying on the fungal cell wall’s surface and break it, leading to the denaturation process of membrane protein and ultimately cell death. Similarly, a surface regularity defect in NPs also inhibits bacterial growth and damages cells membranes of microbial cells and also some organelles of the cell (Gupta et al., 2017; Ray et al., 2024). Moreover, several functional groups from the Morchella conica mushroom extract are attached to produce capped IONPs, which are important for inhibiting microbial strains. This study has correlation with (Dharmaratne et al.,2018; Ansari et al., 2017) in term of anti-bacterial potential of IONPs.

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Fig. 5.

Antibacterial potential of as-prepared biogenic IONPs form Morchella conica.

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3.6 Antifungal activity

To find out the result of IONPs different fungal strains were used to evaluate the fungicidal ability. The three different fungal strains used were Candida albicans (FCBP: 478), Mucor racemosus (FCBP: 0300), and Aspergillus flavus (FCBP: 0064). To find out the antifungal potential of IONPs a range of doses (47–1500 μg/mL) were applied to several fungal strains. Among fungal strains, Aspergillus flavus was the least sensitive (188 ± 4.8μg/mL), followed by Candida albicans (MIC: 94 μg/mL), and the most susceptible fungal strain was Mucor.racemosus (MIC: 47μg/ml) (n = 3). One-way ANOVA showed significant differences in MIC values between bacterial and fungal strains (Ray et al., 2024). Amp-B drug was used as a control, and the ZI was compared with IONPs, and the results have been shown in Fig. 6 and Table 1. Generally, a dose-dependent response was recorded for IONPs. Previous research has reported substantial concentration-mediated fungicidal potencies employing several fungal strains and declared the interaction of IONPs with spores and fungal hyphae, guiding inhibition of their growth (Iqbal et al., 2020; Qadri et al., 2023). These are consistent with our present Morchella conica-IONPs study.

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Fig. 6.

Antifungal potential of as-prepared biogenic IONPs from Morchella conica.

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3.7 Anticancer activity

Different microbes cause different infections in humans and animals, which are life-threatening. With limited treatment methods available, cancer is globally the leading cause of death. Therefore, there is need for techniques to combat multidrug-resistant bacteria and treat cancer cells using various therapeutic approaches (Catalano et al., 2022). To find out the potential of IONPs, the HepG2 cell line was selected. To quantify the control (cisplatin) and IONP values on fast-growing commercial He-G2 cell lines, a dose range of 3.91-500 μg/mL was applied (Fig. 7). The experimental compound exhibited a dose-dependent inhibition of cell viability. At 500 μg/mL, the cells’ morphology was greatly affected, and after CV staining, the number of attached cells was almost negligible. When the dose was reduced to 7.82 μg/mL, the cell morphology remained rather the same, with only a few stained cells observed. Correlated information was adopted from Gharibkandi et al., 2023 and Manimaran et al., 2024. Anticancer drug (cisplatin) and IONPs were compared with one another. The IC₅₀ value of the cisplatin was 15.32 μg/mL, and IC₅₀ for IONPs was 41.81 μg/mL. Morchella conica-synthesized IONPs showed potential against cancer cell lines. Further decreasing the concentration showed that the number of surviving cells increased, and the cells also started to change their shape from circular to elongated. The drug targeting approach of IONPs for cancer therapy has been exposed by (Zhi et al., 2020; Kamal et al., 2023). IONPs and Cisplatin work by producing reactive oxygen species (ROS), which damage lipids, proteins, and DNA (Sanati et al., 2022). This medication causes intra-strand and inter-strand cross links, which result in strand breakage, by binding to the purine bases of DNA. Additionally, NADPH oxidase (NOX), which is activated by cisplatin converts oxygen into hydrogen peroxide (H₂O₂) and superoxide radicals (Sulashvili et al., 2024).

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Fig. 7.

Anticancer potential of as-prepared Morchella conica mediated by IONPs.

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3.8 Antioxidant potential of IONPs

The antioxidant potential of IONPs fabricate by using Morchella conica mushroom extracts (Fig. 8). Antioxidant activity was assessed by using different from 4 to 500 μg/mL. the activity showed concentration dependent tendency with the lowest activity observed at 4 μg/mL (approximately 20%) and the highest activity recoded at 500 μg/mL up to 98% (Yap et al., 2021). Mushroom extract plays a dual role in this route by stabilizing and reducing various iron ions during NP fabrication. To clarify the role of reductive compounds contributing to antioxidant performance, DPPH analysis was carried out by (Sousa et al., 2023). The conclusion, the antioxidant competence of the synthesized IONPs diminished with decreasing the concentration of NPs (Nayak et al., 2024). During the process, it was explored that the highest antioxidant capacities correspond to maximum concentration tested, confirming strong DPPH scavenging at 500 μg/mL. The improved stability and reduction competence of the IONPs may be recognized to varied antioxidant constituents obviously present in the Morchella conica mushroom extract. The dissimilarity and differences from previously reported research may be attributed to a number of significant aspects such as experimental conditions, nanoparticle manufacturing, mushrooms used and size of nanoparticles (Sridhar and Charles, 2019; Iqbal et al., 2019; Yap et al., 2021).

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Fig. 8.

Antioxidant potential of asynthesized Morchella conica mediated IONPs.

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3.9 Brine shrimp cytotoxicity assay

The cytotoxicity of as-prepared IONPs was evaluated using the brine shrimp lethality assay (BSLA). For assessing any naturally occurring compound’s biological potential, this test is the most suitable. Green produced IONPs were studied for their cytotoxicity potential utilizing brine shrimps as an experimental organism. Fig. 9 shows the percentage mortalities of biogenic IONPs at different concentration or doses (4-500 μg/mL) (Hnamte et al., 2020). The results indicated a dose-dependent response. As the IONPs concentration increases the cytotoxic capability also increases. The IC₅₀value was measured as 32.65 μg/mL for IONPs. Vincristine sulphate with IC₅₀ value (4.991 μg/mL) was utilized as a positive control. The cytotoxic potential of IONPs was confirmed by these studies. Our test sample was determined to be in the general cytotoxic category. A comparable result was confirmed by Iqbal et al., 2021 and Muhammad et al., 2022.

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Fig. 9.

Cytotoxic potential of Morchella conica asynthesized IONPs against brine shrimps.

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