Nano-Sized Iron Sulfide: Structure, Synthesis, Properties, and Biomedical Applications

Nano-sized iron sulfides have attracted intense research interest due to the variety of their types, structures, and physicochemical properties. In particular, nano-sized iron sulfides exhibit enzyme-like activity by mimicking natural enzymes that depend on an iron-sulfur cluster as cofactor, extending their potential for applications in biomedicine. The present review principally summarizes the synthesis, properties and applications in biomedical fields, demonstrating that nano-sized iron sulfides have considerable potential for improving human health and quality of life.


INTRODUCTION
With the development of nanotechnology (Li et al., 2019), nanomaterials have become a major resource for the development of novel therapeutic medicines and technologies designed to improve human health and the quality of life (Zhang and Webster, 2009;Esmaeili et al., 2020;Wang et al., 2020). In particular, due to their multiple functionality and excellent biocompatibility, ironbased nanomaterials are frequently used in the biomedical field, such as bioseparation, biosensors, magnetic resonance imaging (MRI), tumor hyperthermia, and drug delivery (Chen and Gu, 2017). In addition, recent studies have revealed that these nanomaterials have intrinsic enzyme-like properties (Gao et al., 2007;Xie et al., 2012;Xu et al., 2018), an important form of nanozyme representing a new generation of artificial enzyme (Wei and Wang, 2013;Dong et al., 2019;Liang and Yan, 2019). Currently, the majority of iron-based nanomaterials are iron oxide which possess excellent supraparamagnetic properties, with catalytic activity mimicking that of oxidoreductases, including peroxidase, catalase, superoxide dismutase, and oxidase (Gao et al., 2007;Liang and Yan, 2019). However, iron sulfide nanomaterials have not been comprehensively studied or used in the biomedical fields. Since O and S are congeneric elements, iron sulfide demonstrates similar physiochemical properties as iron oxide (Fu et al., 2019). In addition, the phases of iron sulfide in nature include mackinawite (FeS), pyrrhotite (Fe 1−x S), pyrite (FeS 2 ), and greigite (Fe 3 S 4 ), etc., which exhibit more variability than iron oxide containing only Fe 2 O 3 and Fe 3 O 4 . The band gap in iron sulfide is smaller than that of iron oxide, leading to the former having more appropriate electron transfer and conductivity (Wadia et al., 2009;Jin et al., 2017;Zhang et al., 2018). Importantly, iron-sulfur clusters are important cofactors in many enzymes which serve as active centers for electron transfer in catalytic processes and respiratory chain reactions (Qi and Cowan, 2011). Therefore, it is anticipated that iron sulfide nanomaterials will display multiple functionalities and they have great potential in biomedical applications. Herein, we will summarize the types, synthesis and properties of iron sulfide nanomaterials and emphasize their applications in biomedical and medical fields. This will provide a comprehensive understanding of iron sulfide nanomaterials and illustrate their considerable potential as novel multifunctional biomaterials in biomedical applications.

TYPE AND STRUCTURE OF IRON SULFIDE
Solid phases of iron sulfides principally comprise FeS (mackinawite), Fe 1−x S (pyrrhotite), FeS 2p (pyrite), FeS 2m (marcasite), Fe 3 S 4 (greigite), and Fe 9 S 11 (smythite). The content of iron within a biomaterial therefore influences its phase, shape, and physical and chemical properties. FeS naturally has a tetragonal structure, with each iron atom coordinated to four sulfurs. For Fe 1−x S, a monoclinic hexagonal is present. FeS 2p forms stable iron (II) disulfides with cubic structures. FeS 2m differs from FeS 2p as an orthorhombic metastable iron (II) disulfide, whilst Fe 3 S 4 is a cubic metastable Fe (II) Fe (III) sulfide. Hexagonal Fe 9 S 11 is related to the Fe 1−x S phase (Rickard and Luther, 2007).
Reported crystal structures of iron sulfide are displayed in Figure 1 (Fleet, 1971;Argueta-Figueroa et al., 2017). FeS possesses a tetragonal layered structure in which the iron atoms are linked through tetrahedral coordination to four equidistant sulfur atoms. A single iron atom is coordinated to four equidistant sulfur atoms. The distance of Fe-Fe is 2.5967 Å. In addition, Fe-Fe bonding is substantial in FeS. To assess the effects of van der Waals forces resulting from the S atoms, sheets including Fe are stacked along the C-axis. The spacing of these layers is 5 Å. The structure of Fe 2 S 2 is closed to FeS. The structure of FeS 2 is similar to that of NaCl in which S 2− is located at the center of a cube. The cubic structure has a low symmetry. In addition, FeS 2 exhibits chirality through absorbed organic molecules. Fe 3 S 4 has an inverse spinel structure in which 8 Fe atoms are located at the tetrahedral A-sites and 16 Fe atoms are located at the B-sites of the octahedron. The unit cell of Fe 3 S 4 is 9.876 Å. In addition, the cubic structure of Fe 3 S 4 forms a closely packed array of S molecules linked by smaller Fe units ( Figure 1D). It has been established that the Fe 7 S 8 structure is a hexagonal supercell (Fleet, 1971). The viable distribution of vacancy sites ideal for the base structure of NiAs was observed to describe the structure of Fe 9 S 10 (Elliot, 2010).

SYNTHESIS OF NANO-SIZED IRON SULFIDES
Nano-sized iron sulfide encompasses a range of iron and sulfur compounds. Firstly, the range of chemical and biological methods for their production are discussed. In addition, the most reported synthesized methods for creating different phases of iron sulfide are presented in Table 1.

Hydrothermal Synthesis
Thermal decomposition is the most commonly-used hydrothermal reaction for iron sulfide production. The typical solvothermal synthesis method of nFeS firstly involves the dissolution of FeCl 3 ·6H 2 O in 40 mL of ethylene glycol. NaOAc and organosulfur compounds (allyl methyl sulfide, diallyl sulfide, diallyl trisulfide, diallyl disulfide, cysteine, cystine, glutathione (GSH), or methionine) are then added under continuous and vigorous stirring. The system was then sonicated for 10 min and transferred to a Teflon-lined stainless steel autoclave. The mixture was reacted at 200 • C for 12 h and precipitates washed three times with ethanol and water. Finally, the products were dried at 60 • C for 3 h (Xu et al., 2018). For FeS 2 , the single source molecular precursor Fe 3+ diethyl dithiophosphate forms an aqueous solution through the reaction of FeCl 3 and (C 2 H 5 O) 2 P(S)SNH 4 , with hexadecyltrimethylammonium bromide (CTAB) added as a surfactant and reacted with a single precursor [(C 2 H 5 O) 2 P(S)S] 3 Fe (Wadia et al., 2009). For FeS, FeCl 3 ·6H 2 O was dissolved in ultrapure water with ethanolamine and thiourea added to the solution. After stirring for 25 min, the mixture was added to a Teflon lined autoclave and reacted at 180 • C for 12 h. The synthesis of Fe 3 S 4 differs from that of FeS. FeCl 3 ·6H 2 O, ethylene glycol, thiourea and H 2 O 2 were mixed and reacted at 180 • C for 18 h in the presence of the capping agent polyvinyl pyrrolidone (PVP) to prevent excessive growth and aggregation of the nanoparticles (NPs) (Moore et al., 2019). Fe 1−x S single crystals were then synthesized through a hydrothermal method by the addition of K 0.8 Fe 1.6 S 2 crystals, Fe powder, NaOH, and thiourea in deionized water, which was reacted at 120 • C for 3-4 days (Guo et al., 2017). Ionic liquids that form extended hydrogen bond systems were then used to form higher structures in the base of the hydrothermal process, as reported by Zheng and colleagues when changing the structure of Fe 3 S 4 (Ma et al., 2010). Generally speaking, the product obtained by the hydrothermal method has better dispersibility and controllability, but iron oxide impurities can also appear during the synthesis of iron sulfide. Meanwhile, multiphase iron sulfide appears to occur easily, as assessed by X-ray diffraction (XRD) patterns for hydrothermally-synthesized samples.

Microwave Production
The principal advantages of microwave-assisted methods, compared with conventional heating include its reduced reaction time, smaller particle size distribution, and higher purity. Ethylene glycol is a solvent suitable for microwave-assisted methods owing to its relatively high dipole moment. For FeS 2 microspherolites, FeSO 4 ·7H 2 O, PVP-K30 and S powder in ethylene glycol can be reacted by microwave in an N 2 atmosphere (Li M. et al., 2011). Although this emerging technique may be more desirable it should be noted that the aggregation phenomenon does not appear to be improved.

Co-precipitation
Chemical co-precipitation does not introduce impurities. The operation is performed under mild conditions and is typically synthesized using Fe 3 S 4 methods, in which iron (II) sulfate heptahydrate and sodium sulfide are dissolved in ultrapure deionized water. The solution was then added dropwise to acetic acid to adjust the pH to 3.0, followed by stirring for several minutes. The reaction was prepared under an N 2 atmosphere (Chang et al., 2011). In addition, green synthesis was achieved in a continuous stirred-tank reactor (Simeonidis et al., 2016). As previously reported, the conditions of synthesis required by coprecipitation are harsher than those of other methods, and the products obtained may show poor homogeneity.

High Temperature Chemical Synthesis
Chemical synthesis methods using high temperatures have been reported for FeS 2 . Briefly, iron (II) acetylacetonate (Fe(acac) 2 ), trioctylphosphine oxide (TOPO) and oleyamine (OLA) were mixed and degassed at 110 • C for 1 h under a vacuum. The mixture was then rapidly heated to 220 • C for 1 h under vigorous magnetic stirring in the presence of nitrogen. Sulfur was then quickly injected into the solution, which was heated to 220 • C for 1 h. Once the solution cooled, ethanol was added to the precipitate to develop the FeS 2 nanoplates (Bi et al., 2011). Synthesis methods for Fe 1−x S and Fe 3 S 4 have also been reported. The rapid injection method has been used to reduce the size of Fe 3 S 4 (Beal et al., 2012). This method of synthesis is highly sensitive to the experimental conditions.

Sonochemical Synthesis
As a convenient and stable synthetic method, Bala and colleagues described specific FeS' sonochemical synthesis. Firstly, sodium sulfide was dissolved in double distilled water. FeSO 4 ·7H 2 O was then independently dissolved in a solution of double distilled water and polyethylene glycol (1:1). The sodium sulfide solution contained a drop of Triton-X surfactant which was added dropwise to the above solution while being continuously sonicated for 30 min. PVP was then added and the system was mixed via ultrasound for a further 30 min (Ahuja et al., 2019).

Other Chemical Methods
Some unusual synthetic chemical methods have been reported. The low temperature synthesis of FeS 2 nanoparticles was described in 2014 (Srivastava et al., 2014a). Briefly, FeCl 3 and sodium polysulfide (Na 2 S x ) were mixed in pH 5.6 acetate buffer in an anaerobic environment. The black solution then was reacted in a 90-100 • C oil bath for 4 h to produce a grayish FeS 2 product. Flux methods were then used to synthesize 1D Fe 7 S 8 . The reaction was conducted within a furnace at 750-850 • C (Kong et al., 2005). FeS can also be created in a biological system (Mei and Ma, 2013).

Biomineralization
The bio-synthesis of iron sulfide using microorganisms is superior for biomedical applications . When microorganisms interact with target ions, they are transported into microbial cells to form NPs in the presence of specific enzymes. In addition to the advantages of green synthesis, biological methods improve the biocompatibility of iron sulfide. Hydrothermal synthesis Nanowires 0.289 Yao et al., 2013 High temperature chemical synthesis Nanosheets 5,000 500 (thickness) Wang et al., 2013 The particles generated have higher catalytic reactivity and a greater surface area. Previous studies have reported that FeS 2 , Fe 3 S 4 , and FeS NPs can be produced by microorganisms. For the biomineralization of Fe 3 S 4 and FeS 2 , a magnetotactic bacterium has been described (Mann et al., 1990). In 1995, FeS materials were produced by sulfate-reducing bacteria grown on iron containing substrates (Watson et al., 1995). Bazylinski and colleagues also reported the formation of Fe 3 S 4 using noncultured magnetotactic bacteria (Lefèvre et al., 2010). Sulfatereducing bacteria were able to produce Fe 1−x S as reported by Charnock and colleagues (Watson et al., 2000). NPs were subsequently formed at the surface by the microorganisms and as such, the porous structure of the iron sulfide NPs failed to prevent normal metabolism. These studies verified the utility of this method for efficient NP production. Chemical biomineralization methods have also been used to synthesize FeS 2 and FeS Quantum dots (QDs) (Jin et al., 2018;Yang et al., 2020).

Iron Sulfide Modifications
Bare nanocrystal cores have an unstable structure that is prone to photochemical degradation. However, those that are unmodified display higher toxicity. As such, biocompatible moieties are essential as they serve as caps for nanomaterials, including polyethylene glycol (PEG), silica, lactose, citrate, and dextran (Simeonidis et al., 2016;Mofokeng et al., 2017). Previous studies have reported the synthesis, characterization, cytotoxicity and biodistribution of FeS/PEG nanoplates in vivo (Yang et al., 2015). An adsorption-reduction method was used to load silver onto the surface of 3-aminopropyltriethoxysilanemodified 3-aminopropyl triethoxysilane (APTES)-modified Fe 3 S 4 particles, achieving the preparation of magnetic composite nanoparticles of Fe 3 S 4 /Ag (He et al., 2013). However, modifications also led to adverse effects. For example, the modification of CTAB inhibited the growth of nano Fe 3 S 4 (Simeonidis et al., 2016).

Characterization of Iron Sulfide
Detailed characterization is required to confirm the synthesis of iron sulfide. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) can be used to image surface morphology. These represent the most direct and commonly-used methods to assess the microstructure, size, and dispersion of the materials. Differences in the synthetic methods have presented variations in TEM/SEM imaging, including within the same phase. For FeS, nanoplates, spherical nanoparticles, and pomegranate flower-like shapes have been reported. FeS 2 exists as large particles, cubic nanocrystals, monodisperse microspherolites, spherical and hexagonal nanoribbons, and nanorods. Typical images of iron sulfide are shown in Figure 2. Table 1 reports the most common morphology and size of FeS, FeS 2 , Fe 3 S 4 , and Fe 7 S 8 . Lattice spaces are measured to judge the crystallinity of the materials. The reported lattice spaces of the iron sulfides are summarized in Table 1. The composition, content, and structure of a substance can be analyzed, measured, and inferred using Ultraviolet-visible (UV-Vis) spectrometry and the absorption of UV and visible light. According to previous studies, the UV absorption peaks of FeS are 285 nm and 500 nm ( Table 2). Information on the composition of the materials and the structure and/or morphology of the atoms or molecules inside the materials can be obtained by XRD analysis. XRD is the most direct indicator of whether a crystal is pure and so represents a convenient system to analyze synthesized materials. JCPDS cards are used to contrast and analyze unknown crystals.  Table 2. For iron sulfide nanomaterials, the X-ray photoelectron spectroscopy (XPS) of Fe and S are essential. FeS materials are presented in Table 2 (Fe 2p: 2p 3/2 (711.4 eV), 2p 1/2 (724.9 eV), S 2p: 2p 3/2 (161.1 eV), 2p 1/2 (166.0 eV)), FeS 2 (Fe 2p: 2p 3/2 (707.0 eV), 2p 1/2 (720.0 eV), S 2p 2p 3/2 (162.3 eV), 2p 1/2 (163.5 eV)), Fe 3 S 4 (Fe 2p: 2p 3/2 (711.0 eV), S 2p: 2p 3/2 (161.0 eV), 2p 1/2 (162.5 eV)), Fe 7 S 8 (Fe 2p: 2p 3/2 (709.9 eV), (711.6 eV), S 2p: 2p 3/2 (163.5 eV), 2p 1/2 (164.7 eV)). Fourier transform infrared (FTIR) spectroscopy can be used to detect functional groups in a complex mixture and so is therefore essential to the characterization of modified iron sulfide. Previous studies have provided FTIR spectra, showing successful modifications by APTES on the surface of the Fe 3 S 4 nanoparticles (He et al., 2013). Fe 7 S 8 /N-C nanohybrids were prepared for FeMOF and FeMOF-S and analyzed by FTIR (Jin et al., 2019). In addition, energy dispersive spectroscopy (EDS) (Paolella et al., 2011;Ding et al., 2016;Guo et al., 2016), dynamic light scattering (DLS) (He et al., 2013;Ding et al., 2016), Raman spectroscopy (Gan et al., 2016;Guo et al., 2016), selected area electron diffraction (SAED) (Kar and Chaudhuri, 2004), X-ray absorption fine structure (XAFS) (Feng et al., 2013), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) (Jin et al., 2019), nitrogen adsorption-desorption isotherms and pore size distribution (Guo et al., 2016) analysis of iron sulfide have been reported as characterization systems.

CHARACTERISTICS OF NANO-SIZED IRON SULFIDES
In addition to both the physical and chemical properties, characteristics include the structure, solubility, stability, reactivity, magnetic properties, and photothermal properties of the products.

Solubility
The major forms of nano-sized iron sulfides are solid precipitates which have poor solubility in water. However, Rickard et al. revealed that sedimental FeS can dissolve at c (S 2− ) ≤ 10 −5.7 M to form Fe 2+ . The solubility can be increased in an alkaline as opposed to neutral environment (Rickard, 2006). FeS does not dissolve in HCl, meaning it cannot be removed with HCl (Rickard and Luther, 2007). According to previous studies, the K sp of FeS 2 was 10 −16.4 at 25 • C. The solubility products of various iron sulfides were assessed and resulted in consensus values for the pKs (FeS: 3.6 ± 0.2; FeS 2 16.4 ± 1.2; Fe 3 S 4 4.4 ± 0.1; Fe 7 S 8 5.1 ± 0.1). This improved our understanding of the solubility of iron sulfide in both synthetic and natural water at room temperature (Davison, 1991).

Stability
FeS is stable within the P-T range of the Martian core (Kavner et al., 2001). Understanding the relationship between the stability of iron sulfide and its chemical environment is of key importance. Once iron sulfide is formed, its structure is reversible. Studies have examined the stability of FeS 2 in different temperatures and the concentrations of absorbed water on the surface. Temperature has little effect on the morphology of FeS 2 under low absorbed water concentrations. However, at above 90 K, the conversion from an octahedral structure to a cubic shape is promoted. At higher concentrations of water, the dependence on temperature is more apparent (Barnard and Russo, 2009). The latter study established that functions of the surface ligands affect the stability of FeS 2 (FeS 2 nanorods synthesized in laboratory) (Barand and Russo, 2009). The stability of FeS contributed to low energy excitation from Fe d to S-Sσ * p . Fe 3 S 4 was observed at 200 • C for 30 h then it transformed to FeS 2 over time (Gao et al., 2015).

Reactivity
Iron sulfide is highly reactive to N 2 and H 2 S. This reaction occurs at room temperature and the adsorption of N 2 is dependent on the surface FeS and on the electronic state of N 2 . A decrease in absorbed N 2 and H 2 S could be explained by the formation of ammonia (Kasting, 1993). The reactivity of Fe 7 S 8 is similar to FeS (Niño et al., 2018). The presence of both Au 1+ and Au atoms has been observed on the surface of FeS 2 . Au deposition increased at higher pH and temperatures. The reactivity of Au 1+ sulfides with FeS 2 have also been investigated (Scaini et al., 1998 (Usher et al., 2005). Recently, FeS 2 was shown to be a potential nanomaterial for prebiotic chemistry due to its highly reactive surface that drives amino acid adsorption (Ganbaatar et al., 2016). Among the most common probes, water molecules have been used to explore the reactivity of FeS 2 (De Leeuw et al., 2000). The different phases of iron sulfide display a wide range of reactivities to chlorinated solvents. Conditions including pH, sulfide concentrations, metal ions, and natural organic matter can affect the reaction kinetics of the degradation of chlorinated solvents (He et al., 2015). The interaction of FeS, Fe 3 S 4 and CO 2 have also been reported. The charge transfer on FeS can also effectively activate CO 2 , whilst Fe 3 S 4 is unreactive to CO 2 (SantosCarballal et al., 2017).

Magnetic Properties
Nanomaterials with magnetic properties have numerous applications, including magnetocaloric therapies, as MRI agents, magnetic separation materials, and magnetic carriers. The discovery of their magnetic properties led to the identification of iron sulfide phases. The ferromagnetism of Fe 7 S 8 can be explained by Fe 3+ ions with excess sulfur (Yosida, 1951). The magnetic susceptibility χ of natural FeS 2 was found to be 64 × 10 −6 68 × 10 −6 cm 3 /moles between 4.2 and 380 K (Mohindar and Jagadeesh, 1979). Magnetic ordering in FeS was inferred and used to prove strong itinerant spin fluctuations. FeS can also be used as a superconductor (Kwon et al., 2011). Even when the structure of FeS is changed from troilite to the MnP-type under high pressure, the antiferromagnetic properties are preserved until the monoclinic structure is formed (Ono, 2007). The magnetic moment then disappears and yetragonal-phase FeS (Tc: 5K) was observed for the same structure as the superconductor FeSe (Tc: 8 K) (Kuhn et al., 2016). Fe 3 S 4 displays high Mrs/χ, (Mrs/χ: the saturation isothermal remnant magnetization: magnetic susceptibility) and its MrsMs (hysteresis ratios) and Bcr/Bc are 0.5 and 1.5 (Ms: saturation magnetization; Bc: the coercive force; Bcr: the coercivity of remanence). Fe 3 S 4 also displays unique high-temperature properties, with a clear drop in magnetization from 270 to 350 • C (Roberts, 1995). Synthesized Fe 3 S 4 contains various crystals from small superparamagnetic grains (non-remanence) to large multi-domain grains (Snowball, 1991). The magnetic hysteresis properties of Fe 7 S 8 have also been studied (Menyeh and O'Reilly, 1997). The relationship between structure and magnetic properties has been reported within variable temperatures. Magnetic transitions occurred within the transformation of the structure (Powell et al., 2004). The magnetocaloric conversion ability of Fe 3 S 4 nanoparticles has been measured under an alternating magnetic field (AMF). Meanwhile, the excellent physical and chemical properties provide magnetothermal thrombolytic ability in medical applications (Fu et al., 2019; Figure 3).

Photothermal Properties
Photothermal therapy (PTT) has attracted considerable attention in recent years. The mechanism of PTT results mainly from photo-absorbing nanomaterials that generate heat through continuous laser irradiation, destroying cancer cells, but causing no damage to healthy tissue. It is therefore necessary to pay attention to the photothermal properties of iron sulfide. PEGylated FeS (FeS-PEG) nanoplates exhibit high near infrared (NIR) absorbance. Using Infrared (IR) thermal imaging, the SCHEME 1 | Schematic diagram of biomedical applications of nano-sized iron sulfide. temperature can reach 70 • C within 5 min. Meanwhile, FeS-PEG displays stronger photothermal conversion efficiency than other known iron oxides (Yang et al., 2015). Ultrasmall FeS 2 nanodots have also been synthesized and have been shown to be useful for photodynamic therapy. Chlorin e6 (Ce6) was used to conjugate FeS 2 in the presence of the template bovine serum albumin (BSA). FeS 2 @BSA-Ce6 nanodots (7 nm) demonstrated good results in in vivo photoacoustic (PA) imaging, MRI and enhanced cellular uptake (Jin et al., 2018; Figure 3).

Biomedical Applications
To-date, a variety of biomedical applications of iron sulfide (catalysts, antibacterial agents, cancer therapies, drug delivery systems, thrombolytic agents, biosensors, antifungal agents, seed improvers in phyto-applications) and their functional mechanisms have been summarized (Scheme 1).

Enzyme-Like Catalysis
Iron sulfur clusters are critical cofactors in many enzymes and proteins which conduct redox reactions and regulate oxidative stress. Thus, nano-sized iron sulfides are expected to perform similar catalysis and act as nanozymes. Previous studies have shown that iron sulfide can effectively activate persulfate (PS) or peroxymonosulfate (PMS) to generate sulfate radicals (Xiao et al., 2020). Other common reactions involving iron sulfide are shown in section Sonochemical Synthesis. Since iron oxide nanoparticles were shown to possess intrinsic peroxidase activity in 2007 (Gao et al., 2007), it has been speculated that iron sulfide has similar properties. The catalytic processes of iron sulfide are shown in Figure 4. High catalytic activity, multi-enzymes activities, harsh environmental resistance, storage stability, and the intrinsic advantages of nanomaterials provide further possibilities for biomedicine development, meaning that good alternatives to natural enzymes exist. The enzymatic activity of iron sulfide has been intensely investigated. In 2010, FeS nanosheets were reported to possess peroxidase-like activity. FeS suspensions were shown to catalyze the oxidation of peroxidase substrates, 3, 3 ′ , 5, 5 ′ -Tetramethylbenzidine (TMB) to produce a blue product in the presence of H 2 O 2 (Dai et al., 2009) (Figure 4). Fe 7 S 8 nanowires' also possessing intrinsic peroxidase activity was also reported in 2013 for which catalytic behavior was observed. The apparent K M of Fe 7 S 8 with TMB as a substrate was 0.548 mM, almost six times lower than that of horseradish peroxidase (HRP). These results demonstrate that Fe 7 S 8 has a higher affinity to TMB than to HRP (Yao et al., 2013). In 2015, magnetic Fe 3 S 4 NPs was shown to possess peroxidaselike activity. Through investigating steady state kinetics, Fe 3 S 4 was shown to possess a higher affinity for H 2 O 2 than HRP. The reasons could be that Fe 3 S 4 binds to and reacts with H 2 O 2 , following which nanozyme is released prior to reacting with the second substrate TMB (Ding et al., 2016). nFeS (Fe 1−x S and Fe 3 S 4 ) (detailed description in section Hydrothermal Synthesis) have been shown to possess both peroxidase-like and catalaselike activity (Figure 4). nFeS is able to decompose H 2 O 2 into free radicals and O 2 , promoting the release of polysulfanes (Xu et al., 2018). FeS 2 has also been shown to possess amylase-like properties (Srivastava et al., 2014b).

Antibacterial Alternatives
Schoonen et al. reported on the antibacterial action of FeS 2 . Its mechanism was shown to be related to the formation of H 2 O 2 . The same group also reported how minerals can induce the formation of reactive oxygen species Schoonen et al., 2006). The rapid absorption of Fe 2+ can influence bacterial metabolism. The oxidization of Fe 2+ to Fe 3+ leads to reactive oxygen species (ROS) production and biomolecular damage. As a result, iron sulfide can act as antimicrobial agents. Iron sulfides have been reported to be effective in bacterial infections. In 2013, He et al. reported on the bacteriostatic activity of Fe 3 S 4 /Ag against E. coli (86.2%) and S. aureus (90.6%). Meanwhile, Fe 3 S 4 NPs without Ag have no effect (He et al., 2013). The Arenas-Arrocena group synthesized Fe x−1 S NPs and reported their antibacterial and cytotoxic activity in 2018. Antibacterial activity against S. aureus, E. coli and E. faecalis was observed (Argueta-Figueroa et al., 2018). In addition, Gao and coworkers discovered antibacterial inorganic iron polysulfides materials that were converted by organosulfur compounds in 2018. Inorganic nano-sulfides (nFeS, Fe 1−x S, and Fe 3 S 4 ) have shown inhibition against Pseudomonas aeruginosa and Staphylococcus aureus. These studies also described new strategies to synthesize iron sulfide nanomaterials. Furthermore, the S. mutans biofilmrelated infections could be prevented by nFeS (Xu et al., 2018) ( Figure 5). The antibacterial properties of FeS 2 -Bi 2 O 3 against the pathogenic microorganisms Mycobacterium tuberculosis and Salmonella have also been measured (Manafi et al., 2019). Diksha et al. reported that FeS NPs could enhance intrabacterial ROS levels in bacteria by light irradiation in 2020. This was revealed   as the primary antibacterial mechanism of FeS NPs (Agnihotri et al., 2020).

Cancer Therapy
Chen and colleagues reported that tumors in mice could disturb iron metabolism in the major organs. The chemical form of iron in the tumors was ferrous-sulfide-like iron and ferritin, highlighting the potential of iron sulfide for cancer treatment . Chang et al. synthesized Fe 3 S 4 particles with magnetic properties through co-precipitation. The NPs were used for cancer hyperthermia providing a new avenue for multimodal anticancer therapies (Chang et al., 2011). In 2015, Yang et al. concluded that triangle nanoplates (FeS/PEG nanostructures) could be used as nanoagents for in vivo MRIguided photothermal cancer treatment. High doses of FeS nanoplates were shown to be safe and effective in mice. This study highlighted the potential clinical use of FeS, for MRI in addition to PTT (Figure 6) (Yang et al., 2015). In 2018, Fe 3 S 4 nanosheets were shown to possess high efficiencies for MRI guided photothermal and chemodynamic synergistic therapy, opening up a new direction for the design of inorganic iron sulfide for future clinical applications (Guan et al., 2018). Tiny nano-sized iron sulfide with simple biomineralization method has also been attention owing to its huge potential in vivo application especially in cancer therapy combined with its excellent photothermal and magnetic performance. FeS 2 @BSA-Ce6 (detailed in section Biomineralization) exhibited good results whether in vivo multimodal imaging or in vivo combined therapy (Jin et al., 2018). Meanwhile, the latest literature proved 3 nm FeS@BSA QDs could be used as T1-weighted MRI contrast agents. Moreover, the ultrasmall QDs showed good results in photothermal therapy and they could be cleared via glomerular filtration into bladder after treatment . The use of iron chalcogenides has also been investigated. Cu 5 FeS 4 -PEG NPs were effective in dual-modal imaging and PTT (Zhao et al., 2016). In 2017, CuFeS 2 nanoplates were used for in vivo photothermal/photoacoustic imaging and cancer chemotherapy/PTT .

Drug Delivery
Iron sulfide holds utility as a drug carrier. In previous studies, modified β-cyclodextrin (β-CD) and PEG Fe 3 S 4 (GMNCs) were used as drug loading NPs. Both β-CD and PEG have been used to control the shape and size of GMNCs as surfactants. In addition, the biocompatibility of Fe 3 S 4 is enhanced, with entrapment efficiencies of 58.7% for the modified delivery of the chemotherapeutic drug doxorubicin. Meanwhile, the enhanced chemotherapeutic treatment of mouse tumors was obtained through the intravenous injection of doxorubicin (Dox) loaded GMNCs (Feng et al., 2013 ; Figure 7).

Thrombolytic Agents
In studies of vascular disease, the removal of thrombosis through non-invasive methods is challenging. However, studies regarding iron sulfide NPs as thrombolytic agents have been reported. Ge et al. first highlighted the utility of Fe 3 S 4 NPs as thrombolytic agents with both photothermal and magnetothermal thrombolytic capability. Using Fe 3 S 4 nanoparticles, celiac vein thrombosis could be prevented using magnetic hyperthermia combined PTT. Both in vivo and in vitro, Fe 3 S 4 has demonstrated beneficial effects for the removal of thrombi (Figure 8), providing a novel hyperthermia strategy for the prevention of thrombosis (Fu et al., 2019).

Biosensors
Iron sulfides have been used as glucose sensors due to their intrinsic peroxidase-like activity (Dai et al., 2009). Glucose sensors can be developed using colorimetric methods in which cascade reactions form the core mechanism of glucose detection. TMB could be oxidized to oxTMB in the presence of glucose, GOx and iron sulfide. H 2 O 2 produced by the decomposition of glucose in the presence of glucose oxidase can be used as a substrate for iron sulfide. Iron sulfide peroxidase-like mimics can oxidize TMB to oxTMB in the presence of H 2 O 2 . Fe 7 S 8 nanowires also possess peroxidase activity. Using a linear range, glucose concentrations of 5 × 10 −6 to 5 × 10 −4 M could be detected (Yao et al., 2013). In 2016, Xian and colleagues used Fe 3 S 4 magnetic nanoparticles (MNPs) to quantify glucose concentrations in human serum. A linear range was measured from 2 to 100 µM, and the limit of detection (LOD) was 0.16 µM (Ding et al., 2016). These studies highlighted the potential of as-prepared iron sulfide as both glucose sensors and artificial peroxidase nanozymes.

Antifungal Agents
In vitro antifungal FeS NPs exhibited significant anti-fungal activity against F. verticillioides at 18 µg ml −1 , with a higher efficiency than standard fungicides (carbendazim (median effective dose (ED50): 230 µg ml −1 ). These were the first reports highlighting the antifungal activity of iron sulfide. The influence of FeS on both seed health and quality parameters of rice was also evaluated based on this antifungal activity. Iron sulfides were shown to be effective in iron deficient soils as an alternative to high dose organic fungicides (Ahuja et al., 2019).

Seed Improvements in Phyto-Applications
FeS 2 represents a photovoltaic material, which increases plant biomass in the seeds of chickpeas (Cicer arietinum). Meanwhile, the mechanism of functional FeS 2 is attributed to its amylase-like activity. In the presence of H 2 O and FeS 2 , starch in the seeds can be broken down to H 2 O 2 , which participates in the absorption of CO 2 and improves plant health. Spinach seeds treated with FeS 2 exhibited broader leaf morphologies, larger leaf numbers and an increased biomass (Srivastava et al., 2014b) (Figure 9). In later studies, seed priming with FeS 2 was reported as an innovative strategy (Das et al., 2016). FeS 2 also improved both seed yield and growth in the Brassica juncea field (Rawat et al., 2017).

CONCLUSIONS
In summary, we have highlighted the most recent methods of nano-iron sulfide synthesis, including nano iron sulfide modifications and characterizations. Strikingly, nano-sized iron sulfides demonstrate versatile physiochemical properties, enzyme-like catalysis, high stability and biocompatibility, which facilitate their biomedical applications. A range of nano-iron sulfides have been assessed in catalysis, tumor therapy, antibacterials and antifungals, drug delivery, biosensors, thrombus removal and in plants. Their advantages include (1) high biocompatibility due to the key role of iron and sulfur in natural life; (2) the photothermal and magnetic properties of nano-sized iron sulfide; (3) their nanostructure and large surface area that can improve drug delivery; and (4) their enzyme-like properties as nanozymes, including their high reactivity to numerous chemical substances that can regulate hydrogen peroxide, ROS and various catalytic reactions to treat related diseases.
Although it has been shown that nano-sized iron sulfides represent great potential in numerous applications in biomedicine (Scheme 2), a number of issues remain to be addressed, including the synthesis of iron sulfide that is stable and in a single phase, with modifications to adapt to the biological environment. Studies have found that the modification of molecular CTAB inhibits the preparation of Fe 3 S 4 NPs due to competitive inhibition with Na 2 S under acidic conditions, resulting in the formation of non-magnetic iron sulfides and other byproducts. Citrate modified nanoparticles have not sufficient particle spacing due to aggregation effects (Simeonidis et al., 2016). In addition, the biomedical assessments of iron sulfides remain sparse, and their mechanisms of action under physiological conditions are poorly understood. The intrinsic enzyme-like properties of iron sulfide as nanozymes may provide a window to understand its biological effects and potential cytotoxicity in vivo. Taken together, nano-sized iron sulfides possess versatile physiochemical properties and enzyme-like properties, which describe a form of distinctive nanomaterials with great potential for use in biomedical applications.