Enzyme-Like Property (Nanozyme) of Iron Oxide Nanoparticles

Abstract

Iron oxide nanoparticles perform biological activity under physiological conditions. They exhibit enzyme-like properties that catalyze redox reactions mediated by natural enzymes of oxidoreductase and are classified into a typical of nanozymes that are defined as nanomaterials with enzyme-like activities. In addition, iron oxide nanoparticles widely exist in biological system, such as magnetosome and ferritin that not only regulate iron metabolism, but also regulate ROS homostasis. The enzyme-like properties of iron oxide nanoparticles render them with broad biomedical applications including immunoassay, biosensor, antimicrobial, anti-tumor, antioxidant. Taken together, iron oxide nanoparticles are bioactive materials and may perform particular biological function in life activity.

1. Introduction

Iron oxide nanoparticles are of typical nanomaterials which can be synthesized using chemical methods or by made from iron oxide minerals. Iron oxide nanoparticles usually include several phases such as magnetite (Fe₃O₄), maghemite (γ-Fe₂O₃), and ferrihydrite (Fh) [1, 2, 3]. The surface structure, crystal phase and facet, shape as well as size dependence make them have various functions [4, 5].

Due to excellent magnetic property, iron oxide nanoparticles have been broadly used in biomedical field, such as magnetic separation of biosamples (nucleic acids, proteins, cells), drug delivery, tumor hyperthermia. Among these applications, iron oxide nanoparticles are assumed as biological inert, even if they have been used as Fenton catalysts for advanced oxidation in waste treatment. In recent years, enzyme-like properties of iron oxide nanoparticles have drawn more attention. In 2007, Yan group found that these nanoparticles performed intrinsic peroxidase (POD)-like activity [6]. Since then, many nanoparticles are found with enzyme-like properties, which boost the development of nanozymes [7, 8]. In particular, iron oxide nanoparticles are found with multiple enzyme-like activities. For instance, Xiaolan Huang found that iron oxide nanoparticles exhibit phosphatase activity that can hydrolyze phosphate ester. Currently, iron oxide nanoparticles are classified as one typical nanozymes and extend their biomedical applications such as antibacterial, antiviral, antitumor, antioxidant, immune regulation.

In addition, iron oxide nanoparticles are also found in biological system, such as magnetosomes of bacteria and ferritin, which are classified as natural nanozymes. These biological iron oxide nanoparticles may be involved in metabolic processes and contribute to life evolution. Thus, the studies on enzyme-like property of iron oxide nanoparticles not only have important significance in extending their biomedical applications, but also provide clues for origin of life. In this chapter, we will summarize the cutting-edged progress in the field of iron oxide nanoparticles related to biological properties (mainly for enzyme-like catalysis) and highlight the significance of such properties in biomedicine and nanobiology.

2. Enzyme-like activities of iron oxide nanoparticles

Iron oxide nanoparticles perform enzyme-like activities that can catalyze the biochemical reactions mediated by natural enzymes under mild condition. The fundamental rationale is that iron serves as critical cofactor in the active center of many natural enzymes. Currently, more than 80 types of natural enzymes are found with iron as cofactors in the form of hemin, coordinated single iron or di-iron, iron-sulfur clusters, which perform the activities ranging from oxidoreductases to nitrogenase. Since iron oxide nanoparticles are rich in ferrous and ferric iron, it is rational to speculate that these nanoparticles mimic the activities of iron-containing enzymes.

It should be noted that the structure of iron oxide nanoparticles is quite different to that of natural enzymes. It is rigid crystal structure in iron oxide nanoparticles, and the active sites may locate on the surface of the nanoparticle. Such inorganic structure endows iron oxide nanoparticles with superior stability and high activity (multiple active sites in single nanoparticle), which makes them suitable for applications under unfriendly environments for natural enzymes. Below, we will introduce the enzyme-like activities reported in the recent decade, in particular in the view of nanozymes.

2.1 Peroxidase-like activity

In 2007, Yan group for the first time reported that ferromagnetic iron oxide (Fe₃O₄) nanoparticles perform intrinsic peroxidase-like activity that can catalyze colorimetric reaction in the presence of hydrogen peroxide (H₂O₂) under acidic condition (pH 3–6.5) (Figure 1) [6, 9]. In this reaction, H₂O₂ is converted to free radicals (•OH) as intermediate. The catalytic behaviors including substrates, optimal pH, and temperature of iron oxide nanoparticles are all similar as those of horseradish peroxidase (HRP). In particular, enzymatic kinetics assay showed that iron oxide nanoparticles fit Michaelis–Menten equation and follow ping-pong mechanism, which confirmed that iron oxide nanoparticles are mimic of HRP. For nanoparticles with the size at 300 nm in diameter, the catalytic efficiency of a single nanoparticle is comparable with a single HRP molecule. However, this does not mean that iron in the nanoparticle has higher activity than that in HRP. Each HRP molecule only has one active site and one iron. In contrast, there may be multiple active sites on the surface of one iron oxide nanoparticle, strongly related to crystal structures, exposed facets, defects, and chemical modifications (Figure 2).

Figure 1.

Iron oxide nanoparticles with peroxidase-like activity catalyze colorimetric reaction mediated by HRP [6]. Copyright 2007, Springer Nature.

Figure 2.

Diagrams of iron oxide slabs with different crystal structures, exposed facets, defects, and chemical modifications [10]. Copyright 2020, ACS.

Xingfa Gao group used density functional theory calculations to investigate the peroxidase-mimetic mechanisms for a series of iron oxide nanosurfaces [10]. They proposed that the activity of these iron oxide nanoparticles mimicked that of POD by following a three-step mechanism in which chemisorption of H₂O₂ was absorbed onto the surface to form two hydroxyl adsorbates and two subsequent reduction processes to remove the hydroxyl groups from the surface. The POD-like catalyses of all iron oxide surfaces proceeded via almost the same mechanism. The properties of iron oxides tuned the energy barrier heights of reaction steps and thus determined which step to be the rate-determining step, resulting in different catalytic kinetics and activity for the surfaces (Figure 3). These theoretical analyses help to understand the relationship between structure and activity of iron oxide nanoparticles.

Figure 3.

Mechanism and kinetics of POD-like reactions catalyzed by iron oxides as determined from DFT calculations [10]. (a) Proposed mechanism of POD-mimetic catalysis of iron oxide slabs. (b) Relative energy values (in eV) for key intermediates and transition states involved in the catalytic cycles. Copyright 2020, ACS.

Compared with natural enzymes, iron oxide nanoparticles exhibit high stability to non-physiological conditions such as low or high temperature, acidic or basic pH, organic solvents. In addition, the peroxidase-like activity of iron oxide nanoparticles is tunable by adjusting their size, morphology, facets, defects, or surface modifications. Similar to natural enzymes, the activity of iron oxide nanoparticles also can be affected by activators or inhibitors [11]. However, the specific peroxidase-like activity of pure iron oxide nanoparticles is in the range of several U/mg, which is much lower than that of HRP with specific activity >150 U/mg [12]. To enhance the specific activity, iron oxide nanoparticles can by hybridized with other nanovectors to form nanocomplexes. Juewen Liu group used molecular imprinting to modify the surface and achieved selective catalysis for the substrates of TMB and ABTS. They found that introducing charged monomers led to nearly 100-fold specificity for the imprinted substrate over the nonimprinted compared with that of bare Fe₃O₄ [13].

2.2 Catalase-like activity

In addition to peroxidase-like activity, iron oxide nanoparticles were found with catalase-like activity under neutral pH by Gu’s group [14]. Using electron spin resonance spectroscopy, they found that both Fe₃O₄ and γ-Fe₂O₃ nanoparticles decomposed H₂O₂ into hydroxyl radicals under acidic condition (pH < 6.5), showing peroxidase-like activity (Fe₃O₄ > γ-Fe₂O₃). However, H₂O₂ was decomposed into H₂O and O₂ under neutral pH (pH 7.4) condition by the two nanoparticles, demonstrating catalase-like activity. These results indicated that the enzyme-like activities of iron oxide nanoparticles are pH-dependent; that is, peroxidase-like activity is dominant at acidic pH and catalase-like activity is dominant at neutral pH (Figure 4).

Figure 4.

ESR spectra subtraction of spin adduct DMPO/•OH [14]. All mixtures contained zero (control) or IONPs at different concentrations and 50 mM DMPO in (a) 100 mM acetate buffer (pH = 4.8) and (b) 50 mM PBS buffer (pH = 7.4). The reaction was initiated by adding 1 mM H₂O₂. Copyright 2012, ACS.

Mover, ferrihydrite, a precursor for most iron oxides, was found with catalase-like activity by Fan’s group [15]. They found that among the 10 forms of iron oxide nanoparticles, 2-line ferrihydrite exhibited the highest catalase-like activity in the pH range of 4.0–8.7, but no peroxidase-like and superoxide dismutase-like activity. The structure-activity studies indicated that the surface iron-associated hydroxyl groups play a key role in catalase-like catalysis. Since natural catalase uses hemin as cofactor in active center, the catalase-like property of the previously mentioned iron oxide nanoparticles may be derived from the iron on the surface (Figure 5).

Figure 5.

The structure-activity relationship of ferrihydrite nanoparticles in catalase-like catalysis [15]. Copyright 2021, Elsevier.

2.3 Superoxide dismutase-like activity

Inspired by some natural superoxide dismutase (SOD) using iron as cofactor, iron oxide nanoparticles are expected to perform SOD-like activity that converts superoxide (O₂^−•) into O₂ and H₂O₂ or H₂O under basic pH (7 ~ 8). However, naked iron oxide nanoparticles exhibited quite low SOD-like activity. Gu et al. modified vitamin B2 on iron oxide nanoparticles and significantly improved the SOD-like activity, providing a reactive oxygen species (ROS)-scavenging ability [16] (Figure 6).

Figure 6.

Modification of VB2 to improve SOD-like activity of iron oxide nanoparticles [16]. Copyright 2020, Springer.

2.4 Oxidase-like activity

Inspired by some natural lipoxidase using iron as cofactor, iron oxide nanoparticles are expected to perform activity inducing lipid peroxidation. Tao Qin et al. incubated iron oxide nanoparticles with liposome at neutral and found that lipid peroxidation occurred by measuring MDA. This phenomenon was repeated using virus containing lipid envelope, which can disrupt viral integrity and degrade surface protein related to infecting host cells [17].

Of noted, although iron oxide nanoparticles exhibited four oxidoreductase-like activities [18], the catalytic efficiency of each activity is different, which may follow the order: peroxidase>catalase>SOD>lipoxidase. In addition, these activities show pH dependency. The pH range may have overlap, and thus, iron oxide nanoparticles may perform multiple activities simultaneously at a specific pH.

2.5 Phosphatase-like activity

Beside the previously mentioned oxidoreductase-like properties, Xiao-Lan Huang discovered that iron oxide nanoparticles exhibited the activity to catalyze the hydrolysis of phosphate ester with enzyme-like kinetics [19, 20]. The iron oxide nanoparticles prepared using a dialysis membrane tube (DMT) system led to the decrease of phosphate esters such as G6P, ATP, G2P and the increase of inorganic orthophosphate (Pi), indicating a catalytic effect on the hydrolysis reaction, which is mediated by natural phosphatase such as purple acid phosphatase (PAP). The authors highlighted that along with other studies of nanozymes such as iron oxide, vanadium pentoxide, and molybdenum trioxide, the oxo-metal bond in the oxide nanoparticles may play critical role for the catalysis in the corresponding natural metalloproteins. In particular, these inorganic nanoparticles with enzyme-like properties not only challenge the traditional concept of enzymes, but also provide new insights into life origin in the early Earth environments (Figure 7) [21, 22].

Figure 7.

The active form of μ-(hydr)oxo iron bridges in purple acid phosphatase (PAP) and different iron oxide phases [19]. Copyright 2018, Mary Ann Liebert, Inc.

3. Iron oxide nanoparticles in biological system

Biogenic iron oxide nanoparticles, such as magnetosome and magnetoferritin, also perform enzyme-like property. Magnetosomes are often synthesized by magnetotactic bacteria species such as Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Nitrospira classes and the candidate phyla Latescibacteria (also known as candidate division WS3) and Omnitrophica (also known as candidate division OP3) of the Planctomycetes–Verrucomicrobia–Chlamydiae (PVC) bacterial superphylum [23]. The biogenic iron oxide nanoparticles have the single-domain size range of 35–120 nm and are covered by bacterial membranes. It was reported that magnetosomes exhibited peroxidase-like activity [24].

Magnetoferritin is caged protein with 24 subunits made up of heavy-chain ferritin (HFn) and light-chain ferritin. Ferritin is spherical, with an outer diameter of 12 nm and interior cavity diameter of 8 nm, in which iron oxide nanoparticles can be formed. It has been found that ferritins are natural nanozymes that exhibit intrinsic enzyme-like activities (e.g., ferroxidase, peroxidase) [25].

4. Broad applications based on bioactivity of iron oxide nanoparticles

The enzyme-like properties significantly extend the application range of iron oxide nanoparticles. Most of the biomedical applications of iron oxide nanoparticles are developed based on their excellent magnetism, such as magnetic separation of proteins, nucleic acids, or cells, hyperthermia of tumor, targeted drug delivery, and MRI contrast. After the discovery of enzyme-like activities, iron oxide nanoparticles have been extensively applied in immune detection, biosensor, antitumor, antibacterial, antiviral, antioxidant, and immune regulation.

First, the peroxidase-like activity allows iron oxide nanoparticles to be applied as an HRP alternative in immunoassays or biosensors for in vitro or in vivo detections [26]. Since iron oxide nanoparticles drive colorimetric reaction of chromogenic substrates, such as TMB, DAB, or ABTS, they can be used in ELISA to replace HRP for signal amplification [6, 27]. In recent year, Yan group has developed a lateral flow test using iron oxide nanoparticles, which is called as nanozyme strip [28]. In this strip, iron oxide nanoparticles are used to replace colloid gold nanoparticles and amplify the signal by their peroxidase-like activity, which significantly improves the detection sensitivity for EBOLA, flu virus, or SARS-COV-2 virus [29]. In addition, iron oxide nanoparticles coupled with natural enzymes such as glucose oxidase can be used for glucose detection via colorimetric reaction or electrochemical detection [30].

Second, owing to oxidoreductase-like activities, iron oxide nanoparticles perform the ability of ROS regulation, which is applied in the treatments of antitumor, antibacterial, antiviral, antioxidant. The peroxidase-like activity boosts ROS generation, which allows iron oxide nanoparticles to be used to kill bacteria [31] or tumor cells [32]. In addition, iron oxide nanoparticles perform antiviral activity by inducing lipid peroxidation in enveloped viruses and subsequently disrupt integrity of virus [17]. Besides generating ROS, iron oxide nanoparticles also can scavenge ROS by utilizing their catalase-like or SOD-like activity. Such unique property can be used for antioxidant treatments in diminishing cytotoxicity [14, 16], ischemia reperfusion of brain [33] and heart [34], neurodegeneration [35]. Recent studies demonstrate that iron oxide nanoparticles can regulate immune system to suppress tumor growth [36] or act as catalytic adjuvant to improve the immune effects of viral vaccine [37].

Besides biomedical applications, iron oxide nanoparticles also can be used with potential in other fields such as environment treatment. By utilizing peroxidase-like activity, iron oxide nanoparticles can be used to detect or degrade the pollutants in environment. For instance, hydrogen peroxide in acid rain can be detected using iron oxide nanoparticles [38]. Pollutants in wastewater, such as phenol, can be degraded by iron oxide nanoparticles [39]. Overall, the enzyme-like activities endow iron oxide nanoparticles with multifunctional property and extend their applications in many important fields.

5. Conclusion

The enzyme-like activities of iron oxide nanoparticles are a unique property for such inorganic nanomaterial. The catalytic types and efficiency are correlated with the nanostructure of iron oxide nanoparticles. Iron oxide nanoparticles act as enzyme mimics of natural enzymes whose active centers are composed of iron as a key cofactor, which not only extend their potential applications, but also indicate that inorganic nanomaterials are not biological inert but active to interact with biological system. These findings may provide a clue for the origin of life from inorganic world to organic and biological world. Though the catalytic efficiency is typically lower than their natural counterparts, iron oxide nanoparticles have high stability and can be scaled up with low cost, thus having a great potential to be used as enzyme mimics (Nanozymes) in many fields.

Acknowledgments

This work was supported by the National Natural Science Foundation of China grant (81930050).

Conflict of interest

The authors declare no conflict of interest.