Iron Oxide Nanoparticles: Green Synthesis and Their Antimicrobial Activity

1. Introduction

Microbial agents such as fungi, bacteria, parasites, and viruses cause various infectious diseases and can be treated with antimicrobial agents. The main concern in treating infectious diseases is that many pathogenic microorganisms have developed resistance against antimicrobial agents, for example, antibiotics [1]. The development of antibiotic resistance is the result of the inappropriate use of antibiotics in community, clinical, and agricultural settings. Consequently, multidrug-resistant bacteria have become a global health threat [2]. Therefore, there is an urgent need to find new effective agents to overcome drug resistance. In this regard, nanotechnology reveals new opportunities for the biosynthesis of nanoparticles with antibacterial properties to solve these challenges [3]. Nanoparticles are materials that show uniqueness in size (generally 1 to 100 nm), structure, physicochemical, electric, magnetic, thermal, mechanical, catalytic, optical scattering properties, and shape [4]. Green synthesis creates metallic nanoparticles through a bottom-up approach that includes a chemical reduction in which a natural product extract replaces a chemical-reducing agent [2]. Iron oxide nanoparticles (IONPs) are defined as “Nanozymes” due to their inherent enzyme-like activities and catalytic properties that are comparable to those of different oxidases such as peroxidases, catalase, sulfite oxidase, and superoxide dismutase [5]. Another feature of these particles is their potential to bind different biological molecules like peptides, enzymes, nucleic acids, lipids, fatty acids, and various metabolites. These nanoparticles have applications in optics, mechanics, biotechnology, engineering, remediation, microbiology, environmental medicine, and electronics, among others [6,7]. This review focuses on green methods for synthesizing iron oxide nanoparticles using bacteria, fungi, yeasts, plant extract, and organic waste. In addition, IONP's prospective antimicrobial applications are discussed in detail. This review will provide compelling information for researchers in the field of nanobiotechnology.

2. Green synthesis of iron oxide nanoparticles

2.1. Synthesis of iron oxide nanoparticles using microorganisms

The synthesis of iron oxide nanoparticles (IONPs) using microorganisms has several advantages over other methods due to the relative abundance of microorganisms, the generation of less toxic by-products for biomedical purposes, and lower cost [8]. Several microorganisms have the ability to accumulate and detoxify metals, and this property can be exploited to convert iron ions from metal salts into nanoparticles [9] using a variety of enzymes, cofactors, proteins, and secondary metabolites of the microorganism [9,10]. These molecules can act not only as reducing agents but also play an important role as stabilizers and protectors of nanoparticles [9]. Microbial proteins containing functional groups, such as hydroxyl, thiol, carboxylic acid, and amine_, have been demonstrated to play a role in the stabilization of metal nanoparticles. These functional groups serve as binding sites for metal ions, which are subsequently reduced to form nanoparticles within the cell wall or the periplasmic space [11].

The synthesis of nanoparticles using microorganisms such as bacteria, fungi, yeasts, and algae can be carried out by intracellular or extracellular mechanisms [10]. The extracellular mechanism consists of the reduction of metal ions, such as iron, by proteins, enzymes, or components of the cell wall of microorganisms [12]. These components work as reducing agents and stabilizers and prevent the agglomeration of iron oxide nanoparticles [11]. For this type of synthesis, the microorganisms are grown under optimal conditions, the supernatant is collected by centrifugation and mixed with an aqueous solution of the metal salt, and the presence of nanoparticles is evidenced by the change in color of the medium [9]. Metal nanoparticles can also be formed when metal ions are trapped on the cell surface by electrostatic interaction and are reduced by enzyme action [11].

Intracellular mechanisms involve enzymatic reduction of metal oxide ions through electrostatic binding of metal ions to -COOH groups of the microbial cell wall, metal ions diffuse into the cell and interact with enzymes to form metal oxide nanoparticles [13,14]. For this type of synthesis, the microorganisms are grown under optimal conditions, then the biomass is washed with sterile water and incubated with a solution of metal salts until a color change indicates the nanoparticle synthesis [9] (Figure 1).

The reduction of metal ions is under the control of several factors, such as functional groups in the cell wall, type of strain, pH, temperature, culture medium, and metal salt concentration. These factors directly or indirectly affect the size, shape, composition, and performance of nanoparticles [15].

2.2. Synthesis of iron oxide nanoparticles using bacteria

The synthesis of iron oxide nanoparticles generally begins with the selection and isolation of bacteria, this includes culturing the strain on specific media such as BHI agar or nutrient agar, followed by incubation under controlled conditions at 35–37 °C for 24–48 h. Additionally, conditions such as carbon dioxide atmosphere may be required. The obtained individual colonies are transferred to the nutrient medium, such as Luria Bertani or nutrient broth, followed by incubation and centrifugation [16]. The supernatant (extracellular method) or the cell pellet (intracellular method) should be taken depending on the synthesis mechanism. The supernatant or sediment suspension is combined with the precursor salts, such as FeCl₃.6H₂O or Fe₂O₃, in the desired volume and concentration. The reaction conditions are optimized by adjusting the pH to the preferred range (5–9), and the mixture is then incubated at different temperatures depending on the strain of bacteria used. After incubation, the color of the medium changes, for example, from dark red to dark brown, indicating the production of IONP’s nanoparticles [17]. When using the intracellular approach, an additional step is required to remove the nanoparticles from inside the cell; for this, a heat shock must be performed by resuspending the cells in buffer, centrifuging, and then using liquid nitrogen followed by a steam bath at 37 °C. The supernatant is centrifuged and collected as a cytoplasmic extract [9,18]. Finally, the synthesized IONPs are dried and characterized. The synthesis of IONPs via different bacterial species is summarized in Table 1.

2.3. Synthesis of iron oxide nanoparticles using fungi

The production of iron oxide nanoparticles via fungal synthesis is considered an attractive option due to its simplicity of scaling, the low cost of the materials necessary for growth, the capacity to produce high amounts of biomass even at an industrial scale, and the low toxicity of the residue [41,42]. In addition, fungal species have higher metal tolerance and bioaccumulation properties, facilitating the synthesis of metal nanoparticles [43]. Fungi are known to secrete large amounts of proteins, enzymes (i.e reductase enzymes [44]), and metabolites that can convert metal salts into various metal nanoparticles [13,45]. Researchers discovered that chitin, a key component of the fungal wall system, is involved in the formation of heavy metal complexes, which are linked to the synthesis of nanoparticles [46]. However, the mechanisms behind metal nanoparticle synthesis using fungi still need to be fully understood.

To optimize the synthesis, some factors must be considered, including the microorganism strain, growth parameters, sample preparation technique, metallic precursor, metallic salt concentration, pH, and reaction temperature [47]. Metal nanoparticles may be synthesized using fungi in a variety of ways, including employing fungal biomass, cell-free filtrate (CFF) from fungal culture, or fungal cell filtrate (FCF) [10]. The use of mycelium is one of the simplest approaches; this process involves culturing the selected fungus strain in an appropriate medium and then separating the mycelium by centrifugation. The mycelium is combined with a metal precursor salt solution and incubated for 4–5 days at 28 °C. The solution is then filtered to produce a colloidal suspension containing the synthesized nanoparticles [22,48]. Nanoparticles can also be synthesized using the cell-free filtrate, which is obtained by growing fungi in a suitable medium and separating the mycelium by filtration. After washing and resuspending the fungal biomass in distilled water, it is incubated on a rotary shaker for 1–5 days, and the filtrate is collected by separating the mycelium with filter paper. Finally, the cell-free filtrate is combined with a solution of iron precursor salts such as ferric chloride and incubated for a few hours on the rotary shaker to achieve IONPs synthesis [45]. For the IONPs synthesis method based on fungal cell filtrate (FFC), the fungal strain is cultivated in the appropriate medium, after which the culture medium is filtered, and the filtrate obtained is centrifuged and filtered again to obtain the final FFC. Finally, a FeCl₃ solution is stirred at room temperature for 5 min. An immediate visual change in color reveals IONPs synthesis [49].

On the other hand, relatively little is known about IONPs biosynthesis from yeast. This type of fungus has a widely varied metabolic and proteomic profile, which means it can produce a wide variety of compounds that have the potential to act as reducing agents [50]. However, more research is required to evaluate its potential and biosynthetic mechanism. Recent reports on the synthesis of IONPs by yeast and fungi are shown in Table 2.

2.4. Synthesis of iron oxide nanoparticles using algae

Algae (microalgae and macroalgae) are commonly used to produce different metallic nanoparticles, and this has become an environmentally friendly option because it is a clean, non-toxic, and sustainable process with the potential to produce a wide variety of sizes, shapes, compositions, and physical and chemical properties [56]. Glycoproteins and different functional groups, such as carbonyl, hydroxyl, and carboxyl, as well as proteins, enzymes, alkaloids, carotenoids, vitamins, terpenes, and cell wall polysaccharides [57,58], are just a few of the biomolecules found in algae. Furthermore, algae can also accumulate heavy metals and produce secondary metabolites with anti-biological fouling effects [56,59]. These characteristics, together with the components they produce, are critical in the reduction, protection, manufacture, and stabilization of [10] nanoparticles [60]. For example, an aqueous extract of Sargassum muticum was used to synthesize cubic iron oxide nanoparticles from FeCl₃ at a temperature of 25 °C, and it was determined that the polysaccharides in the algae extract were the main factor in the reduction of FeCl₃ to Fe₃O₄ nanoparticles with an average size of 18 ± 4 nm [58].

To synthesize nanoparticles from algae, the samples are washed with distilled water and then dried for 72 h under light [58]. The dried samples are then powdered, freeze-dried, weighed, and boiled by mixing the powder with the necessary volume of distilled water. To produce the extract, the boiled extract is subjected to a filtration step. The synthesis of the IONPs begins by combining the extract with a known amount of iron salts, such as FeCl₃ [58,61]. The color change in the media indicates that nanoparticle nucleation has occurred [56,62]. This process is facilitated by the biomolecules found in algae extract, which facilitate the aggregation and self-assembly of nucleonic particles. During this process, the most energetically favorable and stable particle shapes are generated, with cubes, hexagons, pentagons, bars, spheres, triangles, and wires being the most prevalent [63]. Table 3 summarizes information on nanoparticles obtained from various algae extracts.

2.5. Synthesis of iron oxide nanoparticles using plants

Because microorganism-based synthesis requires long incubation periods, extensive research has been conducted on plant-based synthesis of iron oxide nanoparticles since plants, on the other hand, reduce metal salts in a shorter time [9]. Plant extracts used for the biosynthesis of nanoparticles can be obtained from the leaves, flowers, bark, roots, fruits, and stems [10]. Bioactive compounds of these extracts, including mainly tannins, phenols, alkaloids, saponins, organic acids, flavonoids, and vitamins, work as reducing agents for metal salts and stabilizers of nanoparticles [10,71].

Depending on the iron precursor used to form the Fe⁰ metal, electron-rich biomolecules containing hydroxyl groups (-OH) have the ability to effectively reduce iron ions from the divalent or trivalent oxidation state (Fe²⁺ or Fe³⁺) [72,73]. The characteristics of the synthesized nanoparticles are mostly determined by discrete parameters such as plant extract type, volume ratio of the extract, metal salt solutions, and reaction variables such as pH, temperature, and incubation time [72,74].

To obtain the plant extract, the first step is to collect the desired part of the plant of interest, which is then washed with distilled water to remove any contaminants, dried at room temperature, and then ground into a fine powder to provide a good surface for extraction [73]. The active compounds of this powder are extracted by mixing it with the appropriate solvent, either hot or cold; the most frequently employed solvents are water, methanol, ethanol, or aqueous dilutions of these [75,76]. Finally, it is filtered, and the clear solution is mixed with the iron precursor salts (iron chloride, iron nitrate, or iron sulfate) to produce IONPs. In this case, the chemical components in the extract also stabilize the nanoparticles, and the change in color of the solution, which depends on the kind of salt used, intensifies as the nanoparticles are synthesized [7,73,77]. As shown in Table 4, various parts of plants have been studied for the environmentally safe synthesis of iron oxide nanoparticles.

2.6. Green synthesis of iron oxide nanoparticles using from biological waste products

Agro-wastes are abundantly produced during the processing of agricultural products and are frequently discarded into the environment, causing pollution [114]. Agro-waste has been found to be rich in biomolecules such as flavonoids, phenolics, and proteins that can serve as bioreductant agents in the green synthesis of diverse metallic nanoparticles [115]. A new effort to expand the use of agro-wastes as sources of biomolecules in green nanotechnology have received considerable attention, and various agro-wastes have been documented for their relevance in nanobiotechnology [115]. As a result, a range of metallic nanoparticles have been successfully synthesized from agro-industrial wastes, and extracts from these residues have been employed as reducing and stabilizing agents in the majority of cases [116]. The nanoparticles produced in this way have a wide range of activities, including antimicrobial, antioxidant, anticorrosive, and cytotoxic against cancer cells, among others [116,117,118,119]. In general, extracts containing active biomolecules that promote the formation of nanoparticles can be prepared using a simple hot water extraction process of dried and ground agro-waste materials [115]. Information on the nanoparticles obtained from biological waste is highlighted in Table 5.

3. Characterization methods

The green synthesis methods of metallic nanoparticles are continually being improved and optimized to achieve comparable results (size and morphology) to physical and chemical methods. Usually, a change in the color of the reaction mixture indicates the formation of nanoparticles; however, this does not guarantee their formation, so additional processes must be used to identify the particles formed. Continuous UV spectra of the reaction mixture are one of the most frequently applied methods to confirm the formation of nanoparticles [92,132,133] Currently, a variety of characterization methods are useful for evaluating the properties and uses of synthesized nanoparticles, as well as optimizing synthesis procedures [134]. However, no single instrument can characterize all material properties. As a result, numerous complementary and confirmatory techniques for nanoparticle characterization should be applied (Table 6).

The most common nanoparticle parameters studied include size, shape, charge, and surface topology [134]. However, characterizing nanoparticles remains difficult due to the sample preparation procedure, and the characterization technique utilized is directly dependent on the properties required [135].

One of the most important parameters to evaluate is particle size and size distribution, which correlates to the particle's external dimensions. Because this property can determine other properties and the behavior of the final product [136,137], it is important for biological and biomedical applications. This property frequently differs depending on the method employed to evaluate it, which can be attributable to a variety of factors. For example, different methods evaluate different aspects of nanoparticle dimensions, or the method can induce a change in the effective particle size [138]. Among the most common methods for determining the size of iron oxide nanoparticles are: Transmission Electron Microscopy (TEM) [119,139,140,141], Scanning Electron Microscopes (SEM) [71,119,142,143], X-ray Diffraction (XRD) [142,144,145,146], Dynamic Light Scattering (DLS) [147,148,149], High-resolution Transmission Electron Microscopy (HRTEM) [150,151,152], Scanning Transmission Electron Microscopy (STEM) [92].

The physical appearance of a particle (morphology), which comprises shape and surface topography, is important to the behavior of iron oxide nanoparticles. Morphology affects different properties like dispersion, functionality, and toxicity. This property, like the size, is evaluated through direct viewing of the particles, so commonly, electron microscopy techniques are utilized. Among the various forms of iron oxide nanoparticles that have been reported are: round (SEM) [153], spherical (SEM) [142,143], (STEM) [92], (TEM) [146], spheroidal (HRTEM) [152], cubic (SEM) [143], rectangular and triangular (HRTEM) [151], rhombohedral [119], and needle-like (FESEM and HRTEM) [154].

Another important property to determine is the chemical composition of iron oxide nanoparticles. It can be characterized both in terms of surface and bulk chemistry and can be measured at the ensemble or single nanoparticle level. It is mostly determined using spectroscopic techniques, which may be paired with microscopy to attain single-particle level resolution, with the most often used methods being XRD [140,152,155,156], EDS [92,155,157] and EDX [144,158,159,160]. Because the nanoparticle's external layer interacts with the surrounding environment, determining its composition (surface chemistry) is also important and can be obtained using different techniques like Auger electron spectroscopy [161,162], X-ray photoelectron spectroscopy [163,164,165], or inductively coupled plasma mass spectrometry [166].

Along with the chemical composition, another property of importance in the study of IONPs is the crystalline structure of the nanoparticles. In general, XRD is used to determine the three-dimensional arrangement of the atoms in nanoparticles as well as the different phases. However, it has been found that nanoparticles smaller than 5 nm have an impact on the XRD-observed patterns [167]. SAED [168], which is being used increasingly commonly, is a method for a more accurate representation of the crystalline structure of iron oxide nanoparticles [146,149,155].

The charge of nanoparticles in suspension is also an important property to consider. The surface charge is the charge formed on the surface of the nanoparticle as a result of proton adsorption or desorption at hydroxylated sites, and due to the difficulties of directly measuring this charge, the particle's zeta potential is frequently determined. The zeta potential is the difference in potential between the stationary layer of the dispersion medium attached to the dispersed particle and the mobile dispersion medium [168] and can be measured by titration [169] or electrophoresis [92,170,171]. It is important because it is used as an indicator of colloidal stability, which is a critical parameter for many emulsions or dispersions; additionally, it has been used to investigate the interaction of iron oxide nanoparticles with various biological systems, including human cells [172], bacteria [173], and proteins [174].

Nanoparticles' magnetic behavior differs from that of bulk materials, as do other properties. Furthermore, the synthesis method, size, and shape of IONPs all affect the magnetic properties of the nanoparticles. When the particle size is between nano and micro, it exhibits superparamagnetism, resulting in low remanent magnetization and a coercive field. As a result, the nanoparticles can interact with an external magnetic field, but in the absence of this field, the particles exhibit no magnetism. Because of this property, superparamagnetic iron oxide nanoparticles (SPIONs) have a high potential for use in biomedical applications, including controlled drug delivery, chemotherapy, drug formulations, hyperthermia treatment, and radioimmunotherapy, among others [136]. Magnetometry techniques such as VSM (vibrating sample magnetometer) [80,148,158] and SQUID (superconducting quantum interference device) magnetometry [175] are used to determine the net magnetization of nanoparticles.

3.1. UV–VIS spectroscopy

UV–vis spectroscopy, which is based on the Beer-Lambert law, is a common method for investigating the optical and structural properties of nanomaterials, such as shape, size, agglomeration state, concentration, refractive index, and colloidal stability [176,177,178,179,180]. This technique, as previously stated, can be used to confirm the formation of IONPs. Due to their surface plasmon resonance (the resonance of the metal's conduction electrons), metal nanoparticles with a diameter of less than 100 nm efficiently scatter optical light, however, as the particle size increases, the absorption spectra can change, shifting toward longer wavelengths. The nanoparticle's composition, environment, form, and size affect the plasmon resonance's bandwidth, magnitude, and wavelength peak [181]. Greenly synthesized iron oxide nanoparticles typically have absorption spectra with peaks in the 290–370 nm region and an estimated energy band gap from 2.9 to 4.22 eV [82,182,183,184].

3.2. TEM and HRTEM

Transmission electron microscopy (TEM) is a characterization technique used to study the dynamics of physical and chemical processes of the nanostructure at an atomic resolution [185]. An electron beam interacts with the sample, and the transmitted electrons are used to form the image by magnifying and focusing them using an objective lens. The interaction of the electron beam with the sample in the TEM image results in diffraction processes as opposed to absorption processes, which occur in the light microscopic image. Because the sample will be exposed to a high vacuum during the analysis, it must be prepared to endure both the electron beam and the high vacuum chamber [186]. The difference in electron densities of the structures, the density and thickness of the sample, the objective aperture´s diameter, and the energy of the electrons all influence TEM images [187]. Additionally, the contrast image can be generated using either unscattered electrons (light field image) or deflected electrons (dark field image). This technique is widely used to characterize the morphology of nanostructures, such as particle size [188,189], agglomeration state [107], electronic state, and chemical composition [190]. Furthermore, depending on the sample, the electron energies for the beam are generally 100 keV for conventional images and 1 MeV for high-resolution imaging [186]. High-resolution transmission electron microscopy (HRTEM) is a TEM imaging mode that provides atomic-level imaging of a nanoparticle's crystalline structure. With this level of detail, it is feasible to image crystal structures, crystal defects, and individual atoms [181].

3.3. SEM

Scanning Electron Microscopy (SEM) is a non-destructive analytical method used for surface and microstructural analysis that also uses an electron beam, typically ranging from 1 to 30 keV, that scans the surface of the sample. [186,191]. This technique is also useful for determining the purity, homogeneity, and degree of dispersion [192]. When the electron beam strikes a nanoparticle, it emits X-rays as well as three types of electrons: backscattered (or primary) electrons, secondary electrons, and Auger electrons. SEM makes use of both primary and secondary electrons, with the latter being used to generate high-resolution images that reveal features as small as 1-5 nm [192]. An advantage of SEM characterization is that it also helps to study the surface characteristics of the sample of IONPs, given the three-dimensional appearance of the surface structure [100].

Nanoparticles do not require pretreatment since metals conduct electricity when struck by an electron beam, whereas non-metallic materials do. To do this, they are often coated with gold to create a thin coating of conductive material. In standard SEM, however, water molecules evaporate in a vacuum, producing an impediment for the electron beams and obscuring image clarity; hence water removal is important.

3.4. EDX

Energy-dispersive X-ray spectroscopy (EDX) is a technique for determining the purity and elemental composition of synthesized nanoparticles [193]. As previously stated, when an electron beam hits a nanoparticle, X-rays are released along with the three types of electrons, and the energy of the generated X-rays depends on the material under investigation, hence EDX is a technique used in combination with SEM. An image of each element in the sample may be obtained by moving the electron beam over the material, giving an overall mapping of the sample by examining near and at the surface elements and estimating its proportion at different points [181].

3.5. XRD

X-ray diffraction (XRD) is a non-destructive technique used to analyze the crystallographic structure of a material. It can also be used to determine element proportions, the position of atoms in the unit cell, unit cell characteristics, and the number of atoms in the unit cell [194]. The interaction of the X-ray beam with the atomic planes results in partial beam transmission, with the remainder absorbed, refracted, scattered, and diffracted by the sample [181]. High-resolution powder X-ray diffraction (HRXRD) can be used to obtain finer information about the structure of nanoparticles, such as ion spacing and bond length of related substances in single crystal cells, and it is one of the most widely used techniques for determining crystalline perfection and defect studies in single crystals [195], as well as detailed studies of structure and phase transformation in materials [196].

The lattice and structural characteristics may be determined by measuring the diffraction angle obtained when an X-ray beam is incident on the nanoparticles. For example, the crystal size may be determined by applying the original Scherrer equation or a modified version (1).

L= k/βcosθ    or    lnβ=ln k/L+ln 1/cosθ

(1)

Where L = average crystallite size, k = constant related to shape (0.94), λ = wavelength of X-ray, β = peak width of the diffraction peak profile at half maximum height, and θ = Bragg’s angle [197].

3.6. DLS

Dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering, is a frequently employed technique for determining the size distribution profile of NPs in colloidal solutions in the nano- and submicrometer ranges [134]. When a monochromatic laser beam strikes a suspension of tiny particles, the light scatters in all directions, depending on the size and shape of the nanoparticles [198]. Given that the nanoparticles in suspension are in continuous motion due to Brownian motion, the scattering intensity changes over time and offers information on particle size, allowing the hydrodynamic diameter of NPs in solution to be determined. The hydrodynamic diameter is the diameter of the NPs and solvent molecules that diffuse at the same rate as the colloid [199,200]. DLS can also measure the stability of nanoparticles over time, which is useful for understanding how they could behave in different environments and applications [201].

4. Antimicrobial properties of green-mediated synthesis iron oxide nanoparticles

4.1. Antibacterial activity.

Considering the emergence of antibiotic-resistant strains as the leading public health threat worldwide, the search for novel molecules with antibacterial potential is of particular interest. Free iron ions and various metal and metal oxide NPs like silver, ZnO, Al₂O₃, and TiO₂ have been screened as bactericidal or antibacterial agents [202]. However, the main concern when using nanoparticles with antibacterial properties is their high toxicity, which has been reported for ZnO [63, 64]; thus, a nanoparticle that is biologically active against bacteria and biocompatible is needed [61,62]. In this context, IONPs have recently emerged as an alternative due to their bactericide properties and biocompatibility in vivo and in vitro. Despite the potential of nanoparticles as antimicrobial agents, some risks have been reported concerning human health and the environment. In contrast to this, IONPs have been shown to be safe and do not cause toxicity in mammalian cell cultures [203,204], a highly desired characteristic to use in biomedical, clinical, and pharmaceutical applications. The most relevant IONPs with biological activity are Fe₂O₃, magnetite Fe₃O₄, and limonite Fe₂O₃*H₂O [205].

4.1.1. Mechanisms of Antibacterial IONPs Activity.

The primary mechanism by which metal oxide nanoparticles exhibit bactericidal activity is through the regeneration of reactive oxygen species (ROS) [206,207]. ROS species, such a as superoxide radicals (O₂^-), hydroxyl radicals (OH^-), hydrogen peroxide (H ₂ O ₂ ), and singlet oxygen ( ¹ O ₂ ) are involved in this process. It has been proposed that the combined interaction between IONPs and ROS species might be responsible for the bactericidal activity [208]. Furthermore, the interaction of Fe ²⁺ ions and ROS species leads to the formation of free hydroxyl radicals and hydrogen peroxide, ultimately resulting in bacterial death. Various mechanisms of bactericidal action, which have recently been reported, are described below [209].

4.1.2. Membrane disruption

As mentioned, iron oxide nanoparticles can interact with bacterial cell membranes via electrostatic interactions, producing harmful oxidative stress in the bacterium by generating free radicals [210]. Additionally, intracellular oxygen in aerobic bacteria oxidizes zero-valent iron, causing irreversible oxidative damage to the cell. Moreover, because of their small size, IONPs can enter the cell and cause physical damage to the cell wall, limiting cellular growth, replication, and even communication [206].

In E. coli, electron microscope analysis revealed that Fe ₂ O ₃ nanoparticles bind to the cell wall and even penetrate into the cytoplasm, where they accumulate, causing vacuole formation and cell wall disruption [211,212]. Fe ₃ O ₄ nanoparticles bind to the formate hydrogenlyase complex (FHL) found in Gram-negative bacteria cell walls, generating a gradient in the inner membrane that has a strong antimicrobial effect on these bacteria [213]. Another mechanism that causes membrane disruption is disturbing the activity of F0/F1-ATPase, which reduces the membrane redox potential and, as a result, the H ⁺ rate [213]. The size and shape of IONPs have been suggested as key factors in elucidating their bactericidal activity [214].

4.1.3. Protein and DNA damage

ROS damage DNA molecules through their genotoxic action [215]. A decrease in the activity of antioxidant system enzymes (SOD, catalase, and glutathione reductase) might cause an increase in ROS concentration [67]. Metal ions are able to bind the mercapto (-SH), amino (–NH), and carboxyl (–COOH) groups of proteins, including enzymes, causing inactivation or partial inhibition [214]. IONPs can reduce the expression of antibiotic-resistance genes (ARGs) in antibiotic-resistant bacteria found in operating rooms [205]. The capacity of nanoparticles with small sizes to inhibit DNA replication by inactivating topoisomerase has also been reported [216]. ROS interacts with thiol groups in membrane proteins, generating oxidative stress, which denatures the protein, resulting in membrane impermeability and, finally, microbial death [22].

4.1.4. Antibiofilm activities

The physicochemical properties of nanoparticles, such as surface charge and hydrophobicity, are important when evaluating their antibiofilm activity because they influence their capacity to adsorb and penetrate biofilms [217,218]. Positively charged and neutral Fe ₃ O ₄ nanoparticles have been shown to reduce biofilm formation in Streptococcus mutans, the primary cause of caries, as compared to negatively charged IONPs [219]. It has also been observed that IONPs decreases alginate production, which is a key component of the exopolysaccharide of P. aeruginosa biofilm. This reduction is dose-dependent at concentrations ranging from 0.8 to 64 μg/mL [220]. Superparamagnetic iron oxide nanoparticles (SPIONs) cause cell death and biofilm destruction due to vibration damage, local hyperthermia, and ROS generation [221]. These factors lead to the dissociation of bacteria from a biofilm, damage to the bacterial cell wall, membrane rupture, the fusion of different cells with each other, and death [222].

A list of relevant bacteria in the medical field that are susceptible to IONPs is presented in Table 7.

4.2. Antifungal activity

Iron oxide nanoparticles are a promising and safe antifungal agent with many applications, including medicinal, agricultural, and environmental applications [235]. Due to the benefits of sustainability, environmental considerations, and less toxic waste production, green synthesis of nanoparticles used for biomedical applications is gaining popularity over chemically synthesized agents. Research on the antifungal activity of IONPs derived from green synthesis is still limited, although results against a wide range of pathogenic fungi are promising [236] (see also Table 8).

For instance, IONPs (mixed Fe₂O₃ and Fe₃O₄) synthesized using tannic acid in a green method showed antimycotic activity against Trichothecium roseum, Cladosporium herbarum, Penicillium chrysogenum, Alternaria alternata, and Aspergillus niger [236]. The antifungal activity was measured by inhibiting spore germination and determining the zone of inhibition (agar well diffusion assay) of fungal pathogens using a concentration of 0.5 mg/ml in both assays, with an inhibition range of 87.74% – 70.67% and an inhibition zone ranging from 28.67 mm – 18.00 mm, respectively (Table 8). The minimum inhibitory concentration (MIC) varied between 0.063 mg/mL and 0.016 mg/mL for the most sensitive pathogens, Penicillium chrysogenum, and Aspergillus niger, and was comparable with the MIC value shown by the conventional fungicide hexaconazole, which was used as a standard control [236].

Mixed α-Fe₂O₃ and γ-Fe₂O₃ IONPs synthesized from Fe(NO₃)₃·9H₂O, using Platanus orientalis leaf extract in an aqueous phase green synthesis approach also showed promising antifungal activity against Aspergillus niger and even stronger activity against Mucor piriformis, with inhibition zones in the agar well diffusion assay of 16 mm and 26 mm, respectively, using 75 μL of 0.10 mg/mL NPs. These inhibition zones were also equivalent to hexaconazole (Table 8) [105]. The authors attributed the high activity against Mucor piriformi to the fungus's stronger surface interaction with the IONPs.

IONPs generated from FeCl₃.6H₂O with green and black tea leaf extracts were tested against Aspergillus flavus and Aspergillus parasiticus strains and compared with the antifungal activity of copper and silver nanoparticles. Furthermore, their effects on aflatoxin production and adsorption were also evaluated. While silver nanoparticles had greater antimicrobial activity, IONPs displayed the highest adsorption capabilities of aflatoxin B₁, giving them an advantage in applications such as food safety. A concentration of 100 µg/mL IONPs reduced the aflatoxin production by 39.2 – 43.5% in Aspergillus flavus and 47.1 – 51.6% in Aspergillus parasiticus, suggesting significant inhibition of the fungal pathogens (Table 8) [237].

Several other studies have been performed using a variety of biological source materials and green synthesis approaches for IONPs and tested against various fungal strains, which are summarized in Table 8. Briefly, Laurus nobilis L. extracts for IONPs generation results in antifungal activity against Aspergillus flavus and Penicillium spinulosum [3]; leaf extract of Euphorbia helioscopia aids IONPs generation with inhibitory activity against Cladosporium herbarum [238]; and Euphorbia hirta leaf extract can be used to generate IONPs with antifungal activity against Aspergillus fumigatus, Aspergillus niger and Arthogrophis cuboida (Table 8) [94].

It is worth noting that nanoparticles generally allow for surface modification to either change the physicochemical properties or even for targeted drug or bio-cargo delivery via coating/adsorption/binding of therapeutic and bioactive molecules. One such example is chitosan-coated IONPs with antimycotic activity against Fusarium solani, Aspergillus niger, and Candida albicans [239]. While these IONPs were not synthesized in a green approach, the same method used for their modification should be applicable to IONPs from green synthesis. Labeling IONPs from green synthesis with approved fungicidal drugs should also be viable, allowing for more targeted delivery with dual antifungal effects. However, studies using this strategy are currently insufficient, and the safety of such modified IONPs will need to be carefully investigated [240].

The mechanism of action of IONPs for their antifungal activity has not been investigated in detail; however, it is expected to involve the same mechanisms that are responsible for their antibacterial activity. This includes the effects on cell membrane permeability and the generation of radical oxygen species (ROS) via Fenton reactions or photocatalytic reactions with dissolved oxygen, which cause damage to the cell's DNA, cell wall components, and proteins, resulting in cell growth inhibition and cell death. The nanoparticles' high surface-to-volume ratio allows them to strongly adhere to the cell surface of fungi, while their small size allows them to penetrate and damage the cell wall, directing their effects toward the microbial cells [105]. To fully understand the mechanism of action for the antimycotic activity of IONPs derived from green synthesis, more research will be required, which will also help us better understand the different activity profiles of varying IONPs against different fungal strains.

A summary of the described studies is provided in Table 8 and reveals that the antimicrobial activity of IONPs generated by green synthesis can vary depending on the biological source material and synthesis approach used.

4.3. Antiparasitic activity

IONPs derived either from green or chemical synthesis have been used as antiplasmodial and antileishmanial agents; however, most studies have used chemically synthesized NPs [241,242], and only few reports exist of the use of green synthesized IONPs (Table 9). Furthermore, the IONPs have been used in combination with known antiparasitic drugs (i.e., artesunate, albendazole) to enhance their inhibitory effect [242,243,244]. Nevertheless, to the best of our knowledge, none of the studies have used green synthesized IONPs. A key feature is that IONPs have not shown cytotoxic effects in vitro, making them attractive for pharmaceutical and medical applications [14].

When the antileishmanial activity (IC₅₀) of chemically synthesized IONPs was compared to that of biologically synthesized IONPs from Trigonella foenum-graecum plant extract, no greater difference against both promastigotes and amastigotes of Leishmania tropica was observed, despite the fact that the IC₅₀s of biologically synthesized IONPs were slightly lower. However, an enhanced inhibitory effect was observed under LED exposure [245]. High levels of reactive oxygen species were found to be responsible for parasite DNA damage and membrane integrity loss. Notably, these green synthesized IONPs proved to be biocompatible with human erythrocytes. Overall, the results showed that biologically produced IONPs had promising antiprotozoal agent capabilities [245]. In another study, IONPs synthesized from FeCl₃.6H₂O and Nephrolepis exaltata aqueous extract showed favorable parasite inhibitory activity against Plasmodium at 25 μg/mL (62 % inhibition) when compared to chloroquine control samples (70 % inhibition) at the same concentration [14]; however, the mechanism of action was not described.

In general, these IONPs show encouraging antiparasitic properties. Nevertheless, most studies (although limited) are based on non-green synthesized IONPs. As a result, further research should be done using IONPs obtained through green synthesis to better understand their potential to inhibit parasite growth.

4.4. Antiviral activity

Nanotechnology and its applications have shown great potential in the development of novel diagnostic and therapeutic approaches against viral infections [247]. In particular, literature has reported exciting discoveries regarding the antiviral activity of metal complexes and metal-based nanomaterials [248]. Most of these studies involve both the use of the nanoparticles alone or the coating of nanoparticle surfaces with known antiviral compounds [249,250]. The main antiviral mechanisms observed in metal and metal oxide nanoparticles are: inhibition of viral attachment and uncoating by viral-attachment-protein hijacking, disruption of glycoprotein disulfide bonds, and viral structure denaturation via oxidation (reactive oxygen species or ROS) [4]. However, the study of iron oxide nanoparticles (IONPs) has received little attention as potential antivirals. Chemically synthesized IONPs had antiviral activity against the H1N1 Influenza A virus [251,252]. The use of approximately 4.25 pg of IONPs reduced viral titer by 8-fold while maintaining 50% of cell viability [252]. However, the human and environmental effects of the inherent toxicity associated with laboratory chemical synthesis and its byproducts are a serious issue [253]. A recent study described a moderate inhibition of polio virus-1 and polio virus-2 after treatment of infected L20B (mice cell line transfected with poliovirus receptor) and RD (human rhabdomyosarcoma) cells with IONPs synthesized from the aqueous fruit extracts of Hyphaene thebaica (Table 9) [246]. Overall, the antiviral potential of IONPs is vast and still remains unexplored, which poses a fundamental question to be fulfilled by future studies regarding green-synthesized nanoparticles.

5. Conclusions and future prospects

The present review provides a thorough examination of Iron Oxide Nanoparticles (IONPs), specifically focusing on their synthesis through environmentally sustainable methods, their notable antimicrobial capabilities, and the significance of the most prominent characterization methods. This combination of factors sheds light on both the potential benefits and challenges associated with the utilization of IONPs as antimicrobial agents.

The green synthesis of IONPs, frequently involving the utilization of plant extracts, microorganisms, or biocompatible materials, aligns cohesively with the contemporary emphasis on sustainability and eco-friendly practices within the realm of nanotechnology. Such an environmentally conscious approach not only minimizes the ecological footprint but also engenders the development of nanomaterials that are safer and more biocompatible.

The demonstrated antimicrobial efficacy of IONPs, spanning a broad spectrum of microorganisms encompassing bacteria, fungi, parasites and viruses, represents a promising avenue for addressing the escalating global challenge of antimicrobial resistance. The observed synergistic effects when IONPs are combined with traditional antibiotics present a potential solution for countering resistant strains.

Nevertheless, it is imperative to acknowledge and confront the constraints and apprehensions surrounding the use of IONPs. Cytotoxicity remains a substantial challenge, necessitating meticulous optimization of dosage, surface functionalization, and comprehensive assessments of biocompatibility. Furthermore, the long-term consequences of IONPs exposure and the prospect of their accumulation in biological systems demand further rigorous investigation.

Looking forward, the prospects of IONPs as antimicrobial agents appear promising. Advancements in surface modification techniques, innovative strategies for targeted delivery, and rigorous regulatory approval procedures are poised to unlock their full potential. Additionally, mindful consideration of their potential environmental implications and the implementation of responsible disposal methodologies are integral components of future research and applications.

In essence, the combination of green synthesis methodologies, potent antimicrobial attributes, and rigorous characterization of IONPs has unveiled a promising trajectory for combating microbial infections. While challenges persist, the convergence of research endeavors, accompanied by an unwavering commitment to safety and sustainability, offers a glimpse into a future where IONPs stand poised to revolutionize the landscape of antimicrobial therapeutics, thereby mitigating the escalating global threat of antimicrobial resistance.