Investigation of Biofabricated Iron Oxide Nanoparticles for Antimicrobial and Anticancer Efficiencies

Abstract

Capparis zeylanica leaf extract was employed in this work to create iron oxide nanoparticles (α-Fe₂O₃) using anhydrous ferric chloride. The UV spectrum, XRD, FT-IR, and FE-SEM with EDX methods were used to characterize the fabricated nanoparticles. The iron oxide nanoparticles obtained were spherical in form, with an average crystallite size of 28.17 nm determined by XRD. The agar well diffusion method was used to assess the antimicrobial activity of the α-Fe₂O₃ nanoparticles created in this study against pathogenic organisms, Gram-negative bacteria (Escherichia coli and Pseudomonas aeroginosa), Gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes), and fungi (Candida albicans and Aspergillus niger). Among the pathogens tested, S. pyogenes had the highest zones of inhibition (25 ± 1.26 mm), followed by S. aureus (23 ± 0.8 mm), E. coli (23 ± 2.46 mm), P. aeroginosa (22 ± 1.86 mm), C. albicans (19 ± 2.34 mm) and A. niger (17 ± 3.2 mm). The substance was further tested for anticancer activity against A549 (lung cancer) cells using the MTT assay. The cytotoxic reaction was found to be concentration-dependent. The present study, therefore, came to the conclusion that the bio-effectiveness of the manufactured α-Fe₂O₃ nanoparticles may result in applications in biomedical domains.

1. Introduction

Nanotechnology has developed a reputation in nanoscience during the past twenty years. Researchers and scientists are actively working to create diverse nanoparticles from innovative materials for various uses [1]. One of the most significant transition metals for biological purposes is iron oxide. Hematite (α-Fe₂O₃) is the most stable polymorph of iron oxide and is ideal for biomedical applications due to its adaptable magnetic and optical characteristics, great chemical stability, and biocompatibility. There are a number of ways to make iron oxide nanoparticles, including mechanochemical methods, wire electrical explosion, electrochemical methods, and green synthesis.

Plants are more favoured than microorganisms as biological resources because of their low cost and simple accessibility in nature. In addition to lowering the metal salt, the biomolecules used in the synthesis also functionalize the nanoparticles’ surfaces, leading to synergistic benefits in applications such as cancer therapy or the development of antimicrobial agents [2]. A promising alternative method for creating nanoparticles is through green synthesis. Compared to the currently available, traditional physical and chemical approaches, green synthesis provides a number of benefits [3]. Iron oxides are one of the most biocompatible metal oxide nanoparticles due to their exceptional minuscule physical properties, including superparamagnetism, firmness in liquid solutions, low susceptibility to oxidation, long blood half-lives, and flexible surface chemistry. They have a wide range of applications in environmental regulation, including as antibacterial drugs, in the adsorption of dyes, in food-related processes, and in the biomedical sector (drug delivery, magnetic cells, immunoassays, and hyperthermia treatment of cancer) [4,5,6,7,8,9,10,11]. In the current work, anhydrous ferric chloride was converted into α-Fe₂O₃ nanoparticles using a Capparis zeylanica leaf extract and the nanoparticles were examined for antimicrobial and antiproliferative activities.

2. Materials and Methods

2.1. Materials

Fecl₃.6H₂O of analytical grade was the sole substance employed in the manufacture of the hematite nanoparticles. We bought the bacterial culture medium from HiMedia (Mumbai, India). Sigma Aldrich in the USA provided the ethyl acetate, antibiotic solution, and Dulbecco’s Modified Eagle’s Medium (DMEM).

2.2. Biosynthesis of α-Fe₂O₃ Nanoparticles

The procedure recommended in prior research [12] was slightly modified to prepare the C. zeylanica leaf extract. C. zeylanica leaf extract was added to 50 mL of a 0.1 M aqueous solution of ferric chloride hexahydrate along with leaf extract. The orange-coloured ferric chloride hexahydrate solution changed to a brownish-black colour, precipitating a black substance. After being sonicated for 30 min, the mixture was filtered, rinsed with distilled water, and dried. To produce iron oxide nanoparticles, the dried precipitate was heated for three hours at 600 °C in a muffle furnace.

2.3. Characterization of α-Fe₂O₃ Nanoparticles

The X-ray diffraction (XRD) method was used to study the crystallinity of α-Fe₂O₃ nanoparticles. Using a UV–Vis NIR double-beam spectrophotometer, the optical properties of α-Fe₂O₃ was examined at different wavelengths between 300 and 700 nm. The substituents of the nanoparticles were examined using Fourier-transform infrared (FT-IR) spectroscopy (Perkin Elmer RX-I Spectrophotometer). A field emission scanning electron microscope (FE-SEM) was employed to examine the morphology of the α-Fe₂O₃ nanoparticles. The energy-dispersion X-ray (EDX) method was used to analyse the composition of the α-Fe₂O₃ nanoparticles.

2.4. Antimicrobial Activity

The antibacterial activity of the artificial α-Fe₂O₃ nanoparticles was investigated using ethyl acetate-based leaf extracts from C. zeylanica. The pathogenic organisms were collected from the Microbial Type Culture Collection (MTCC), Chandigarh, India, and included Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The aforementioned cultures were then cultivated and kept alive at 35 °C for bacterial species and 30 °C for fungal species on Mullar-Hinton agar (MHA) and Sabouraud Dextrose Agar (SDA) media, respectively. In MHA and SDA plates, a 6 mm well was pounded. A micropipette was used to inject 20 µL of sample into each plate’s well. Gentamycin (50 µg) served as a positive control for bacteria and fungi. The bacterial and fungal cultures were incubated for 24 h at 30 °C and 72 h at 37 °C, correspondingly. Each experiment was executed three times, with measurements taken in millimetres.

2.5. Anticancer Activity

The National Center for Cell Science (NCCS), Pune, India, provided the lung cancer cell line A549. The cells were cultured in DMEM media supplemented with 10% FBS, Penicillium (100 g/mL), streptomycin (100 g/mL), and amphotericin B (5 g/mL) and incubated overnight. Utilizing the established 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) tests as described by Chelliah and Oh [13], the cytotoxic impact of synthesized α-Fe₂O₃ nanoparticles on the A549 cell line was assessed. A 96 well plate with 2 × 10⁴ cells per well and a volume of 100 µL was used for cell incubation. After 24 h, the cell layer was treated with various doses (1.45, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 g/mL) of Fe₂O₃ nanoparticles. Cells were incubated at 37 °C for 4 h after addition of 50 µL of MTT (1 mg/mL) to each well. The development of Formosan crystals was then disrupted in 200 µL of dimethyl sulphoxide after the medium had been withdrawn. A microplate reader was used to measure the absorption over the course of 15 min at a wavelength of 540 nm

2.6. Statistical Analysis

Each test was run in triplicate. One-way variant analysis was performed, and the results are presented as means ± standard errors.

3. Result and Discussion

3.1. UV–Visible Spectrum of α-Fe₂O₃ Nanoparticles

The spectrum of the α-Fe₂O₃ nanoparticles’ diffuse reflectance in the UV–visible range is displayed in Figure 1a. The prominent absorption peak at 354 nm corresponds to α-Fe₂O₃ nanoparticles [14].

3.2. XRD of α-Fe₂O₃ Nanoparticles

Figure 1b shows the XRD pattern of the α-Fe₂O₃ nanoparticles. The (012), (104), (110), (113), (024), (116), (018), (214), and (300) planes were, respectively, assigned to the XRD peaks at 24.76, 33.52, 35.12, 41.67, 49.79, 54.02, 57.22, 62.52, and 63.82 2h. The wide, sharp peaks further demonstrated the well-crystallized hexagonal structure of the α-Fe₂O₃ nanoparticles (corresponding to JCPDS file no. 33-0664).

3.3. FTIR Spectra of α-Fe₂O₃

The FTIR spectra of α-Fe₂O₃ are shown in Figure 1c. The synthetic α-Fe₂O₃ nanoparticles’ infrared spectra (FTIR), which were recorded in the 400–4000 cm⁻¹ wavenumber region, were used to identify the compound’s chemical bonds and functional groups. The existence of an absorbed water molecule was indicated by the wide band at 3407 cm⁻¹, which was attributed to the O-H stretching vibration [15]. The peaks at 3216 cm⁻¹ in the fluoride-treated ferrous oxide-hydroxide nanoparticles were somewhat displaced to lower frequencies, which may have been the result of fluoride adsorption, ion exchange, or both [16]. The bending vibrations of the C-H bond in the organic compounds were thought to be responsible for the absorption peaks at 2967 cm⁻¹ [17]. These adsorption peaks at 2076 cm⁻¹ for the N=C=S (iso-thiocyanate) group provided evidence that proteins and other bioactive substances were present on the surface of the biosynthesized iron nanoparticles [18]. A small peak at 1028 cm⁻¹ was caused by the stretching vibration of C-O H [19]. The FeO₆ octahedron was present in the samples, as seen by the prominent absorption bands at 548 cm⁻¹, which were attributed to the stretching of the Fe-O bond [20].

3.4. FE-SM with EDAX

In Figure 1d, pictures of the synthesized α-Fe₂O₃ nanoparticles obtained using FE-SEM are displayed. The α-Fe₂O₃ nanoparticles in the FE-SEM image are distributed uniformly and have a spherical shape. The α-Fe₂O₃ nanoparticles had an average particle size of 28.17 nm. The fabrication of α-Fe₂O₃ nanoparticles is depicted in Figure 1e as a histogram of the particle size distribution. Figure 1f displays the EDAX pattern. The components that made up the resulting α-Fe₂O₃ nanoparticles were iron (Fe) and oxygen (O).

3.5. Antimicrobial Activity

The substance was tested for its antimicrobial effectiveness against harmful organisms. Streptococcus pyogenes exhibited the largest inhibitory zones (25 mm), followed by Staphylococcus aureus (23 mm), Escherichia coli (23 mm), Pseudomonas aeroginosa (22 mm), Candida albicans (19 mm), and Aspergillus niger (17 mm). With the use of the agar well diffusion technique, a considerable zone of inhibition (ZOI) was discovered, as shown in Figure 2a.

Table 1. Antimicrobial activity of biosynthesised α-Fe₂O₃ nanoparticles against microorganisms.

S. No.	Name of the Organism	Zone of Inhibition (µg/mL−1) a
		PE	Fecl3.6H2O	α-Fe2O3	Positive Control	Negative Control
1.	Streptococcus pyogenes	9 ± 1.37	14 ± 1.26	25 ± 2.56	20 ± 1.32	0
2.	Staphylococcus aureus	7 ± 1.24	12 ± 1.43	23 ± 2.23	17 ± 1.55	0
4.	Escherichia coli	6 ± 0.76	7 ± 0.20	23 ± 1.34	13 ± 1.22	0
3.	Pseudomonas aeruginosa	8 ± 1.23	10 ± 1.21	22 ± 1.23	15 ± 1.35	0
5.	Candida albicans	7 ± 1.19	11 ± 0.17	19 ± 0.98	11 ± 1.12	0
6.	Aspergillus niger	5 ± 0.99	6 ± 1.27	17 ± 1.62	9 ± 0.75	0

^a Mean values of three triplicates ± standard deviation. PE—plant extract, Fecl₃.6H₂O—ferric chloride hexahydrate, α-Fe₂O₃—biosynthesized iron oxide nanoparticles, Control—ethyl acetate.

shows that the rate of microbiological growth decreased as the α-Fe₂O₃ nanoparticle concentration was increased. The results for the synthesized nanoparticles’ antimicrobial efficacy against our chosen microorganisms showed that they possessed strong antimicrobial activity.

Similarly, Leisha et al. [21] demonstrated that biosynthesized iron-oxide nanoparticles can inhibit the growth of Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Streptococcus mutans and have potential bactericidal activity. The strong antimicrobial activity of the synthesized nanoparticles was likely brought about by the presence of the plant extract on their surfaces. Even though these were extremely positive results, additional research is required to determine whether iron-oxide nanoparticles are a viable alternative to silver nanoparticles for the treatment of bacterial infections. Although the precise mechanism of the antimicrobial action of the α-Fe₃O₄ nanoparticles that causes damage to bacterial proteins and DNA has not yet been identified, it was speculated that it may involve oxidative stress brought about by reactive oxygen species, such superoxide radicals (O₂^-), singlet oxygen (¹O₂), hydroxyl radicals (-OH), or hydrogen peroxide (H₂O₂). The process that produces ROS can readily diffuse into the cytoplasm of the cell, causing ROS-induced release in the mitochondria and causing death [22].

3.6. Anticancer Activity

The α-Fe₂O₃ nanoparticles were tested for cytotoxicity against human A549 cancer cells at a range of doses (1–100 mg/mL). A phase-contrast inverted microscope was used to examine the cell growth. After receiving treatment with α-Fe₂O₃ nanoparticles, A549 cells shrank and their nuclei became fractured and compacted, as shown in the microscopy images in Figure 2b. At lower hematite concentrations (1–10 mg/mL), significant morphological alterations were not seen. According to our research, the substance has a concentration-dependent cytotoxic effect on A549 lung cancer cells (Figure 2c). At a concentration of 15.6 µg/mL, which is regarded as the IC50 value, α-Fe₂O₃ nanoparticles demonstrated 50% cell killing. According to the designated activity levels of the US National Cancer Institute’s Center For Cancer Research, the produced nanoparticles are moderately active [23].

4. Conclusions

Biofabricated α-Fe₂O₃ nanoparticles were successfully synthesized using C. zeylanica leaf extract. The conformations of the nanoparticles were characterized using various techniques. The α-Fe₂O₃ nanoparticles possess admirable antimicrobial and antiproliferative activities.