Catalytic Mechanisms of Nanozymes and their Applications in

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Catalytic Mechanisms of Nanozymes and their Applications in Biomedicine Haijiao Dong, Yaoyao Fan, Wei Zhang, Ning Gu, and Yu Zhang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00171 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Bioconjugate Chemistry

Catalytic Mechanisms of Nanozymes and their Applications in Biomedicine Haijiao Dong1†, Yaoyao Fan1†, Wei Zhang1,2, Ning Gu1,*, Yu Zhang1,* 1

School of Biological Science and Medical Engineering, Southeast University, State Key Laboratory of

Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, Nanjing, Jiangsu 210096, China 2 The

Jiangsu Province Research Institute for Clinical Medicine, the First Affiliated Hospital of Nanjing

Medical University, Nanjing 210029, P.R. China

ABSTRACT: The research on nanozymes has increased dramatically in recent years and a new interdiscipline, nanozymology, has emerged. A variety of nanomaterials have been designed to mimic the characteristics of natural enzymes, which connects an important bridge between nanotechnology and biological science. Unlike natural enzymes, the nanoscale properties of nanozymes endow them the potential to regulate their enzymatic-like activity from different perspectives. The mechanisms behind those methods are intriguing. In this review, we introduce these mechanisms from the aspects of surface chemistry, surface modification, molecular imprinting and hybridization and then focus attention on some specific catalytic mechanisms of several representative nanozymes. The applications of nanozymes ranging from bioassay, imaging to disease therapy are also discussed in detail to prove the fact that the inherent physicochemical properties of nanomaterials not only make nanozymes the analogues of biological enzymes, but also endow them incomparable advantages and broad prospects in biomedical fields. Finally, four characteristics and some challenges of nanozymes are summarized.

1. INTRODUCTION Nanozymes, inorganic nanostructures with intrinsic enzymatic activities, are emerging as novel artificial enzymes that have increasingly attracted researchers’ great interests in the past decade. An accidental discovery that Fe3O4 nanoparticles (NPs) possess intrinsic peroxidase (POD)-like activity was first reported by Yan et al. in 2007.1 Shortly thereafter, numerous nanomaterials have been discovered that have POD-,

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catalase (CAT)-, superoxide dismutase (SOD)- or oxidase (OXD)-like catalytic activities. With intensive research efforts focused on nanozymes, nanozymology, an emerging interdisciplinary field connecting nanotechnology and biology, has gradually formed. Compared with natural enzymes, nanozymes exhibit several advantages such as facile preparation, low cost, high catalytic efficiency and stability against denaturing. In addition, the nanoscale properties of nanozymes allow tunability to their enzymatic activity. Thus, it is possible to ameliorate their specificity and catalytic activity accordingly through synthesis optimization, change in size and morphology, chemical doping or surface modification, etc. Owing to these fascinating features, nanozymes serve as nanoreagents have been widely used in various applications such as biosensing, immunoassay, disease treatment and environmental engineering. Although there have been many strategies using various materials to prepare nanozymes for different applications, the catalytic mechanisms behind these methods are rarely thought-provoking. Due to the interplay between surface chemistry and structure, it is challenging to acquire a basic insight into how these factors affect the catalytic performance of nanozymes. Therefore, from the perspective of pure researches, decoding the catalytic mechanisms behind enzymatic-like reactions is critical in the ingenious design and modulation of nanozymes with improved activity and intelligent multifunction. In this review, we first introduce the general mechanism regulation strategies of nanozymes from the perspective of the factors that influencing the catalytic activity. Then, the specific catalytic mechanisms of several representative nanozymes are emphasized. Moreover, the unique application potentials of nanozymes in biomedicine, ranging from bioassay, imaging to disease therapeutics, are also discussed.

2. MECHANISM REGULATION OF NANOZYMES 2.1 Size- and shape-dependent surface chemistry The size and shape of nanozymes are important parameters that influence their


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enzymatic activity. As a matter of fact, this kind of influence is essentially related to various surface chemistries of nanozymes. Considerable efforts have been made to regulate the size or shape of nanoparticles toward superb catalytic efficiencies in various advanced applications.2-14 The catalytic regulation associated with the surface chemistry will be discussed in detail as below.

2.1.1 Size-dependent surface chemistry Since the chemical reactions occur mainly on the surface of nanozymes, the catalytic activities have great relationship with the amount of catalytically active atoms exposed on the surface, which is usually inversely proportional to the diameter of particles.1, 1518

For example, Yan et al. reported that iron oxide nanoparticles (IONPs) with different

sizes showed different catalytic levels towards 3, 3’, 5, 5’-tetramethylbenzidine (TMB), a typical peroxidase substrate, in the order 30 nm > 150 nm >300 nm.1 The special redox cycle of cerium ion between Ce3+ and Ce4+ contributes to the excellent SOD and CAT catalytic properties of cerium oxide nanoparticles (CeO2).16 As a decrease of CeO2 size, more Ce3+ are formed at the surface due to the lattice strain caused by the presence of oxygen vacancies in a stable fluorite-type structure.14, 16 Thus, the catalytic activity of smaller CeO2 is superior to the larger one. (Figure 1)

Figure 1. Size-dependent catalytic activity of cerium oxide nanoparticle. The SOD- and CAT-like catalytic activities are improved for smaller CeO2 and for particles with more Ce3+ surface concentration. CNP-5, CNP-8, CNP-23 and CNP-28 represent CeO2 with transmission electron microscopy (TEM) sizes of 5 nm, 8 nm, 23 nm and 28 nm, respectively. (Reprinted with permission



from ref 16. Copyright 2018 Royal Society of Chemistry.)

Nonetheless, in certain cases, there could be a converse trend in which the catalytic performances improve along with the increase of size.17 It is believed that sizedependent enzyme activity of nanozymes is also related to the coordination numberdependent chemical environment of surface catalyst atoms.19 The smaller the size, the lower coordination number. The unsaturated coordination of atoms provides a very strong binding to the reactants, intermediate, or product molecules so that it is difficult to couple with other substances on the surface, which reduces the reaction speed.5 This opposing result requires more systematic studies and the underlying mechanism remains to be further explored, although some assumptions have been proposed.

2.1.2 Shape-dependent surface chemistry The selective synthesis of nanozymes with varying specific crystal planes is an important prerequisite for exploring their shape-dependent catalytic activity. Significant progress in synthesis technology has developed many methods to tailor nanoparticles with specific shape.2, 4-8, 12, 13, 20

The influence of shape on nanozymes’ catalytic performance is complex, which can be understood from different perspectives. On the one hand, as commonly recognized by us, the structural differences among various shapes inherently bring about different crystallographic surfaces and surface-to-volume ratio; On the other hand, the surface physicochemical properties of these nanomaterials highly depend on the coordination environment and number of dangling bonds of different crystal plane atoms, which affecting the catalytic activity.

Mugesh et al. synthesized V2O5 nanoparticles with four morphologies to simulate the activity of glutathione peroxidase (GPx), including nanowires (VNw), nanosheets (VSh), nanoflowers (VNf) and nanospheres (VSp) (Figure 2a-b).4 The enzymatic Michaelis– Menten kinetics of them were studied with different concentrations of H2O2 and


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glutathione (GSH). The results showed that the maximum velocity (Vmax) of these four particles followed the order VSp > VNf > VSh > VNw. The mechanism behind this phenomenon was deeply investigated through in situ FT-Raman spectra. It was demonstrated that V-peroxido species resulted from the reaction of V=O species with H2O2 was the predominant intermediate in the catalytic cycle.(Figure 2e) The formation of V-peroxido species was most favored on the surfaces of VSp and the formation rate decreased in the order of VSp ((100) and (010)) >VNf (major (010); minor (001)) >VSh (major (001); minor (010)) > VNW (001). To explain this order, the energy of H2O2 adsorption (ΔE1) and V-peroxido species formation (ΔE2) were obtained by quantum chemical calculations. The sum of ΔE1 and ΔE2 was the total energy change (ΔEtot). Since vanadium (V) coordination saturation in (001) facet, the ΔE1 was lowest and Vperoxido couldn’t form in this facet. (Figure 2f) In contrast, (100) and (010) facet possessed higher ΔE1 due to the unsaturated coordination of the surface V atoms. (Figure 2g-j) What is more, (010) facet possessed the highest ΔEtot. These results consistently indicated that the most reactive surface was (010) facet, whereas the least active was (001) facet, which was consistent with the GPx activity of these four nanoparticles.4



Figure 2. (a-d) Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of (a) VNw, (b) VSh, (c) VNf, and (d) VSp. (e) Schematic diagram of the reaction of GSH and H2O2 on the surface of orthorhombic V2O5 crystal. (f), (g) and (i) ) Most favored orientation for interaction of H2O2 with {001}, {100} and {010} crystal facets, respectively. (h) and (j) V-peroxido intermediate on {100} and {010}, respectively. (Reprinted with permission from ref 4. Copyright 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Weinheim.)

As mentioned above, the coordination environment of surface atoms is an important factor that can be considered in regulating the catalytic activity of nanozymes. In the field of metal catalysts, the dissociation of chemical bonds within reactant molecules is considered to be the rate-limiting step in a catalytic reaction. The lower coordination number of metal atoms, the stronger adsorption energy for reactant, which would decrease the activation barriers of reactant molecules dissociation, thereby accelerating the catalytic event.19 In addition, the uncoordinated metal atoms located at the open surfaces are expected to exhibit higher-energy d states which approximate the highest


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occupied states near the Fermi energy, usually forming antibonding states. Consequently, metal atoms interact strongly with their adsorbates.19

Interestingly, different crystal facets of nanozymes may also exert a great influence on the O2 adsorption and activation process which is known to play an important role in some enzymatic reaction. For instance, in some noble metal nanozymes-catalyzed oxidation reactions, the conversion of electron spin state from inert ground triplet O2 to highly reactive singlet O2 (1O2) is an indispensable step.8,

21

A typical example

experimentally demonstrated that the yield of 1O2 was closely related to the different crystal facets on the surface of Pd nanoparticles.8 1O2 stemmed from the spin-flip process of O2, which occurred at the surface of Pd crystal plane via electron transfer from Pd surfaces to O2. It has been proved that the formation of 1O2 was more preferred on Pd nanocubes enclosed by {100} facets rather than Pd octahedrons by {111} facets. The reason could be concluded that the charge transfer from metal surface to frontier antibonding orbital of O2 on Pd {100} brought about a slightly larger O-O bond and smaller magnetic moment of O2 than that on Pd {111}.8 Thus, it can be seen that crystal plane is a key parameter to alter the chemisorption state of O2, in turn, modulate the catalytic activity of nanozymes.

2.2 Surface coating or modification Since enzyme-like reactions usually occur on the surface of nanozymes, the catalytic performances are closely related to the surface characteristics and the catalytic environment of particles. Generally, the active sites of most metal- and metal oxidebased nanozymes originate from the surface metal atoms, the amount of which is difficult to change after particles synthesis.22 While proper surface coating or modification is an effective and promising method to engineer their activities by regulating surface charge, acidity, exposed active sites or product desorption and so on.23 Three aspects are mainly introduced as following: ion adsorption, small molecule modification and macromolecular coating



2.2.1 Ions adsorption As commonly recognized by us, the combination of surface atoms and substrates to form intermediate complexes is an important prerequisite for a catalytic reaction. This reminds us that the enzyme-like activity of nanozymes could be adjusted by its affinity for substrates. Ions adsorption is one of the effectives strategies.

For example, the surface charge of CeO2 can be reversed from positive to negative due to the adsorption of F- or SO42- which increases the affinity for cationic substrate TMB and facilitates the activity of CeO2.24, 25 Besides, the adsorption of F- can also reduce the surface energy of particles, thereby reducing the affinity towards oxidation products and promoting the products desorption.25 Moreover, similar with F-, SO42- serves as strong electron withdrawing ligand and can promote the adsorption of O2 on CeO2.24 The adsorbed O2 will accelerate the movement of lattice oxygen and redox cycle reaction (Ce3+ = Ce4+ + e-) of CeO2, which boosts the catalytic reaction between O2 and substrates. (Figure 3a) More interestingly, this promotion effect of SO42- selectively occurs on CeO2 nanorods (NRs) ({110}) rather than CeO2 nanopolyhedra (NPO) or nanocubes (NCs). (Figure 3b-e)


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Figure 3. (a) Schematic diagram of OXD-like activity mechanism of CeO2 in the presence of facetselective trigger molecule SO42-. The absorbance changes of TMB at 652 nm before and after adding Na2SO4 using CeO2 (b) nanorods (NRs), (c) nanopolyhedra (NPO), and (d) nanocubes (NCs) as catalyst. (e) The histogram of detailed absorbance difference obtained from figure (b), (c) and (d). (Reprinted with permission from ref 24. Copyright 2017 Elsevier.)

In fact, not all ion adsorption plays a positive role in the enzymatic activity of nanozymes. In biologically relevant media or buffers, there exist some components, such as phosphate ions, which have much affinity for “Ce” in the 3+ oxidation state. This interaction leads to the changes in surface chemistry of cerium nanoparticles (CeNPs) and the formation of cerium phosphate, which block the redox of Ce between 3+/4+, and eventually result in the loss of SOD-like activity. Interestingly, this affinity interaction is selective and rarely occurs on CeNPs (4+).26, 27 What is more, research has also indicated that S2- could react with the surface atom of Au NPs to form sulfides. As a result, the catalytic active sites of AuNPs were covered and then the catalytic



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activity was weakened.28 Besides, the phosphate group could also bind to the surface Fe3+, hindering the reaction of substrates with IONPs.26, 29 But that is not necessarily a bad thing. Given that these changes in enzyme-like activity caused by ion adsorption can be visualized using chromogenic substrate, various colorimetric sensors have been developed to detect the harmful ions in the environment. 2.2.2 Small molecule modification Some small molecules such as amino acids, nucleoside triphosphates (NTPs), dopamine can bind to nanozymes and alter their catalytic performances. Some examples are summarized in Table 1. Given space limitations, only amino acid modification, for example, is discussed in detail below.

Table. 1 Effects of small molecule modification on the activity of nanozymes Modification

Nanozy

Enzyme-

Activity

material

me

like activity

regulation

His

PdNPs

POD

Promotion

Regulatory mechanism ·Better dispersibility and smaller size

Ref 23

·Decreasing binding energy of Pd and promoting substrate activation ·Increasing hydrophilicity His

Fe3O4

POD

NPs

CAT

Promotion

·Modifying with histidine residues to mimic the active sites

30

in natural HRP ·Hydrogen bonds formed between the imidazole group of His and H2O2 can weak the O-H bond for easily split the O-O bond of H2O2 and lead more negative charge of O to enhance this adsorption onto Fe3O4 NPs.

Phe

CeNPs

OXD

Optimizing the

·The formation of hydrogen bonds between D-/L-Phe and

31

DOPA enantiomers

stereoselecti vity ATP ADP

Citrate

POD

Promotion

· ATP may alter the redox state of citrate which

coated

thermodynamically facilitate the oxidation of TMB through

AuNPs

lowering activation energy.

32

·ATP may act as H2O2 activator and electron transport medium. ATP

Fe3O4 NPs

POD

Promotion

·𝐹𝑒IIATP2 ― + 𝐻2 𝑂2→𝐹𝑒III(𝑂𝐻 ― )ATP2 ― + ∙ OH (pH7.4)

(pH 2~11)

· ATP may stabilize the oxidized TMB or ABTS by facilitating single electron transfer reactions.


33

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NTP

Nanocer

OXD

ia

Promotion,

·NTPs serve as “coenzyme” for Nanoceria.

GTP >

·Nanoceria can interact with the nucleic base residues of

ATP >

NTPs and possess phosphatase-like activity.

UTP > CTP

34

·The released energy by hydrolyzing NTP can improve the OXD-like activity of Nanoceria in return.

Catecholamin

Fe3O4

es

NPs

POD

Inhibition

·The covalent binding between the catechol moiety of

35

catecholamines and surface Fe3+ induces the formation of complexes that inhibit the POD-like activity of Fe3O4 NPs.

Melamine

AuNPs

POD

Promotion

·The strong interaction between surface-bond AuCl4-/

36

AuCl2- and amine groups of melamine induces the formation of AuNPs-melamine aggregates, which accelerates the decomposition of H2O2. Triethyl

CeO2

CAT: Inhibition

·TEP can electronically couple with the surface Ce ions and

phosphite

NPs

SOD: Promotion

reduce the surface Ce4+ of CeO2 NPs.

(TEP)

Lan et al. investigated the role of histidine (His) on the POD-like activity of Pd NPs.23 They found that His-modified Pd NPs (His-Pd) had better activity compared with bare Pd NPs. It could be traced to three reasons: firstly, the decoration of His leaded to a better dispersibility and smaller size of Pd NPs, and thus there were more surface-tovolume ratio to interact with reaction substrates. Secondly, the surface chemistry of Pd NPs was changed due to the presence of His. X-ray photoelectron spectroscopy (XPS) showed that the corresponding peaks of Pd 3d in His-Pd shifted to a low binding energy compared with bare Pd NPs. What is more, the proportion of Pd°on the Pd surface was increased. In the catalytic mechanism of POD-like for noble metal nanozymes, metal M°is involved in the activation of H2O2 to release hydroxyl radicals (OH).38, 39 Thirdly, His-Pd had a smaller contact angle to enhance hydrophilicity, which is beneficial to the interaction between nanozymes and substrates.

In particular, appropriate chiral amino acids modification can also optimize the stereoselectivity of nanozymes. Qu et al. constructed chiral OXD-like CeNPs by modifying with D- or L-phenylalanine (Phe).31 3,4-dihydroxyphenylalanine (DOPA) enantiomers were selected as substrates that can be oxidized by CeNPs and thus appear the absorbance at 475 nm. (Figure 4a) Based on UV-Vis absorption experiments, the


37


bare CeNPs were not stereoselective for DOPA. (Figure 4b) However, CeNP@D-Phe showed the highest catalytic ability for oxidizing L-DOPA, while CeNP@L-Phe were more effective towards D-DOPA. (Figure 4c-e) It is related to the number of hydrogen bonds formed by the interaction of DOPA enantiomers and Phe enantiomers. As shown in Figure 4f-i, the molecular docking between phenylalanine (L-Phe/D-Phe) and DOPA (L-DOPA/D-DOPA) was carried out using the Discovery Studio 2.0 program. The results showed that L-DOPA interacted with D-Phe to form three hydrogen bonds, which was more than other combinations. It was excellent agreement with the result that CeNP@D-Phe showed the highest activity for L-DOPA.31

Figure 4. (a) The stereoselective catalytic oxidation of L- or D-DOPA by CeNP@D-Phe or CeNP@L-Phe. The absorbance changes of L- or D-DOPA at 475 nm in the presence of (b) bare CeNP, (c) CeNP@D-Phe and CeNP@L-Phe. Michaelis- Menten plot for the enantiomers of DOPA oxidation by (d) CeNP@D-Phe and (e) CeNP@L-Phe. (f-i) Energy-minimized average interaction models between Phe enantiomers and DOPA enantiomers. (Reprinted with permission from ref 31. Copyright 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

2.2.3 Macromolecular coating Organic polymers, such as PVP, PAA, PEG and PEI, are commonly used surfacemodifying macromolecules. In addition, some biopolymers (such as DNA, proteins) can also be attached to the nanozymes surface via electrostatic interactions,


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coordination or hydrogen bonding. Intriguingly, the regulation effects of macromolecular are contradictory. On the one hand, macromolecular modification can increase the catalytic efficiency of nanozymes by improving their colloidal stability and the affinity with substrates. On the other hand, over-coating or modifying would enclose the active sites or form steric hindrance that hinders the interplay of substrates and nanozymes.22, 40-45

Liu et al. found that DNA modification could increase the POD-like activity of Fe3O4 and CeO2 by changing their surface charge. Conversely, the OXD-like activity of CeO2 was reduced.45 Presumably, these phenomena resulted from the different catalytic mechanisms between OXD- and POD-like activity for CeO2. In OXD-like activity, the surface of particles acts as oxidant and the substrates have to bond to the particles to initiate the catalytic reaction. However, the true oxidant in POD-like activity is free radical produced by H2O2, which can decompose substrates without the combination of the substrates and particles. Therefore, the blocking effect of DNA on surfaces inhibited the OXD-like activity of CeO2, but had little effect on their POD-like activity.

The effects of dextran and polyacrylic acid (PAA) on the OXD-like activity of nanoceria were studied by Perez et al. They found that the oxidation capacity of nanoceria suffered a decline after modifying with these two macromolecules. Noteworthily, the nanoparticles with a thin PAA (1.8 kDa MW) coating showed a higher catalytic activity than those with a thicker dextran (10 kDa MW) coating.44 Thus it can be seen, the coating thickness of polymers is a notable factor affecting the activity of nanozymes. Basically, the thicker coating, the lower enzyme activity.

Appropriate coating can improve the stability, biocompatibility or target ability of nanozymes without affecting their activity, which lays a foundation for the further application in biomedicine.38,

46, 47

For example, Karakoti and Seal et al. modified

cerium oxide nanoparticles (CNPs) with a smaller molecular weight PEG (600 MW), which did not significantly affect the regeneration of Ce3+ and the SOD-like activity of



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CNPs.47 The biocompatibility of PEGylated CNPs was greatly enhanced and this material will offer a promising alternative to some radical scavenger.

More examples about macromolecular coating are summarized in Table 2. In fact, macromolecular modification is a complex and delicate process that is not only related to the materials used and coating thickness, but also closely connected with the size, charge, morphology and composition of nanozymes. Even sometimes different laboratories would acquire conflicting results.48, 49

Table. 2 Effects of macromolecular coating on the activity of nanozymes Activity

Modification

regulation

material DNA

Nanozyme

Enzyme-

Regulation mechanism or purpose

Ref

like activity Fe3O4 NPs

POD

·Changing the surface charge to increase

45

the affinity with substrates DNA

CeO2 NPs

POD


40, 45

the affinity with substrates Promotion

·Improving the dispersibility of CeO2 NPs ·Allowing more active sites to be exposed DNA

AuNPs

POD


RNA

the affinity with substrates

DNA

·Forming steric hindrance

42

45

·Hindering the proximity of substrates Dextran

CeO2 NPs

OXD

PAA

·Covering the active sites

44

· Hindering molecules transfer on the surface of nanocrystals

Inhibition

· The surface charge, molecular size and Protein

hydrophobicity of proteins can affect their Fe3O4 NPs

POD

PEG

interaction with nanoparticles. ·The thicker coating covers the active sites

1

on the surface of nanoparticles.

Dextran ssDNA

43

AuNPs

POD

· Providing a shielding effect against

50

interaction of AuNPs with substrates PEG (600 MW) No

Protein

significant

Dextran

change

BSA

·Increasing the residence time of Nanoceria

SOD

47

nanoparticles inside cells ·Providing biocompatibility

Au clusters

POD

·Improving the robustness


47

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2.3 Surface molecular imprinting In spite of the increasing numbers of methods have been developed to ameliorate the catalytic activity of nanozymes, so far, how to address substrate specificity remains a technical challenge and requires more research efforts. Recently, surface molecular imprinting technology has gradually been used to engineer substrate binding sites on the surface of nanozymes to enhance selectivity.51-53

For example, IONPs, serve as POD mimetic, can catalyze a diverse range of substrates in the presence of H2O2. However, using neutral acrylamide and N-isopropylacrylamide as monomers and imprinting TMB or 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) template molecules on the surface of IONPs could enhance their selectivity to the imprinted molecules. (Figure 5a) Moreover, charged monomers could further improve this effect, nearly 100-fold.51

In order to enhance the selectivity of AuNPs-based glucose oxidase (GOD) mimic, Zhang and Gu et al. employed aminophenylboronic acid (APBA) as the imprinted polymer shells in which glucose molecules (Glu) were imprinted.53 As illustrated in Figure 5b, after eluting Glu, the porous polymer shells with specific binding pockets were capable of specifically recognizing, capturing and enriching Glu, which acquired remarkable selectivity to the substrates. The introduction of oxygen-donating group, heptadecafluoro-n-octyl bromide (PFOB), into the imprinted material could further increase the catalytic efficiency of AuNPs up to 270-fold.53 This enhanced catalytic efficiency might also result from the space confinement effect by the molecularly imprinted porous shells.



Figure 5. Schematic illustration of (a) Fe3O4 NPs-based POD mimic and (b) AuNPs-based GOD mimic constructed by molecular imprinting. (Reprinted with permission from ref 51 and 53. Copyright 2017 American Chemical Society and 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

2.4 Synergistic effect of hybrid nanozymes The catalytic performance depended on single nanomaterials, in many cases, cannot meet the practical applications in various areas. It is highly desired to engineer hybrid nanozymes combining the respective properties of each component to enhance or synergistically exert their catalytic activities. There have been many reports exploring the regulation mechanisms of hybrid nanozymes to mimic the complexity and functionality of natural enzymes.54-65

The doping of Zn caused the lattice structure of CuO NPs with many defects, dislocations and vacancies which were the active sites for the generation of OH radicals.


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This is the reason why the POD-like activity of Zn-CuO was greatly improved compared with pristine CuO.54

A significantly enhanced POD-like activity of Pd-Ir nanocubes was achieved by depositing an ultrathin Ir atomic layer on the surface of Pd nanocubes.55 (Figure 6a, b) It was ascribed to the fact that the adsorption energy of Ir-Pd{100} surface was larger than that of the Pd{100}, resulting in more reactive for adsorbing oxygen-containing species and easier dissociation of H2O2 into OH. However, the POD-like activity of Pd nanocubes with thicker Ir overlayers was lower than that with a single Ir overlayer. Briefly, although highly reactive surfaces could dissociate H2O2 more easily, they hampered the transfer of H from TMB to the surface of substrates.55

Moreover, a growing research interest has been focused on the integration of the respective enzymatic activities of different nanomaterials into a multienzyme collaborative platform. Qu et al. used V2O5 nanowire as GPx mimic while MnO2 NPs as SOD and CAT mimic. Dopamine served as a linker of these two nanomaterials.57 (Figure 6c) V2O5@pDA@MnO2 was obtained to simulate the intracellular antioxidant enzyme-based defense system, which exhibited excellent intracellular reactive oxygen species (ROS) removal effect through the synergistic antioxidative ability.57 (Figure 6d) Furthermore, AuNPs were used, instead of MnO2, to construct a V2O5@pDA@AuNPs nanocomposite. This system could mimic the enzyme cascade reaction of POD and GOD for the detection of glucose and target complementary DNA.59



Figure 6. (a) High-angle annular dark-field scanning TEM (HAADF-STEM) images of Pd-Ir coreshell nanocube. (b) The comparison of catalytic efficiency of horseradish peroxidase, Pd cubes and Pd-Ir cubes. (c) Schematic diagram of the synthesis of V2O5@pDA@MnO2. (d) The intracellular ROS removal progress based on SOD/CAT/GPx-mimicking antioxidant synergetic system. (Reprinted with permission from refs 55 and 57. Copyright 2015 American Chemical Society and 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Some porous carbon structures are often regarded as the promising support materials for nanozymes due to their large surface-to-volume ratio and excellent electron transport capabilities.58, 61, 64, 65 When doping with other catalytic units, it is possible to construct multifunctional carbon-based porous hybrid nanozymes. The existence of the porous, on the one hand, as a nanoreactor, provides more reaction binding sites which facilitate the rapid diffusion of dissolved oxygen. Moreover, the porous channels act the role of spatial confinement. In the mesopores, the aggregation of nanoparticles is effectively reduced and the collision probability between substrates and catalytic units is increased, which is beneficial to the enzyme activity. Usually, Fe, N, Co or the like


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have been doped in the porous carbon structures and serves as the catalytically active center. Moreover, N atoms-doping can also induce heterogeneous charge distribution of carbon atoms, resulting in excellent ability for adsorbing oxygen, thereby enhancing catalytic performance. In addition to porous carbon, mesoporous silica is also used to encapsulate some inorganic nanozymes or natural enzymes to achieve the synergistic effect of catalytic efficiency.56, 62

3. CATALYTIC MECHANISM OF REPRESENTATIVE NANOZYMES 3.1 Metallic compound nanozymes Some transition metal elements have variable oxidation states due to the fact that there are plenty of easily lost single electrons in their electronic configuration. (Table 3) Among the compounds composed of these metal elements, such as Fe3O4, Fe4[Fe(CN)6]3, Co3O4, CeO2, CuO, Mn3O4 and V2O5 NPs, the metal elements can be converted between variable valence states, making them promising candidates for mimetic enzyme. Several metallic compound-based nanozymes and their catalytic mechanisms are discussed in detail below.

Table. 3 Several common oxidation states of some metal elements Elements oxidation states

Fe

Co

Ce

Cu

Mn

Ir

Pt

+Ⅱ

+Ⅱ

+Ⅲ

+Ⅰ

+Ⅱ

+Ⅱ

+Ⅱ

+Ⅲ

+Ⅲ

+Ⅳ

+Ⅱ

+Ⅲ

+Ⅲ

+Ⅲ

+Ⅲ

+Ⅳ

+Ⅳ

+Ⅳ

+Ⅳ

+Ⅵ

+Ⅴ

+Ⅴ

+Ⅶ

+Ⅵ

+Ⅵ

3.1.1 Iron-based nanozymes IONPs (e.g., Fe3O4 and Fe2O3) were first discovered with intrinsic POD-like activity, similar with the Fenton reaction.1, 66 (Equation 1-3) Subsequently, Gu and Zhang et al. verified the pH-dependent POD- and CAT-like activities of IONPs in the IONPs/H2O2 reaction system.67 The detailed mechanisms were explored through electron spin



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resonance spectroscopy (ESR) that was first employed by Yin et al. to demonstrate and quantify the formation of oxygen radical induced by nanomaterials. At pH 4.8, ESR spectra indicated that H2O2 could be catalyzed by Fe2O3 or Fe3O4 to produce high reactive OH, which can be captured by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form spin adducts DMPO/OH and then generate typical four peaks with intensity ratio of 1:2:2:1. This signal intensity was proportional to the concentration of IONPs.67 (Figure 7a) At pH 7.4, the ESR signal of UV-irradiated OH gradually diminished upon the addition of Fe3O4, Fe2O3 and catalase. This signal reduction indicated that IONPs could act as CAT and decompose H2O2 into H2O and O2 in neutral pH.67 (Figure 7b) 𝐹𝑒3 + + 𝐻2𝑂2→𝐹𝑒𝑂𝑂𝐻2 + + 𝐻 +

(1)

𝐹𝑒𝑂𝑂𝐻2 + →𝐹𝑒2 + + H𝑂2·

(2)

𝐹𝑒2 + + 𝐻2𝑂2→𝐹𝑒3 + + 𝑂𝐻 ― + 𝐻𝑂·

(3)

Figure 7. ESR spectra of spin adducts DMPO/OH. (a) The mixtures contained IONPs at different concentrations or zero (control), 50 mM DMPO and 1 mM H2O2 in acetate buffer (100 mM, pH = 4.8). (b) The effect of Fe3O4, Fe2O3 and catalase on the generation of OH in H2O2/UV system. The mixtures contained 25 mM DMPO, 5 mM H2O2 in PBS buffer (50 Mm, pH = 7.4). (Reprinted with permission from ref 67. Copyright 2012 American Chemical Society.)

This pH-dependent catalytic mechanism is inextricably linked to the reversible redox of Fe ion on the surface of IONPs. Ferrous ion (Fe2+), in particular, plays an important role in the activation of H2O2 and the generation of free radicals (e.g., OH 、 O2-


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/HO2).66, 68, 69 Once these highly active radicals are formed, they may react by two competitive reactions: organic oxidation and H2O2 decomposition.69

From the perspective of redox potential, electrons are usually transferred from a low redox potential to the high one. That is the reason why Fe3+/Fe2+ (standard redox potential 0.771 V) cannot directly transfer electrons from TMB (standard redox potential of TMBOx/TMBRe, 1.13 V) to H2O2 (standard redox potential of H2O2/H2O, 1.776 V). Instead, the substrate is oxidized by the radicals generated from the reaction of Fe2+ with H2O2. In the higher pH environment, poor oxidation property of H2O2 is traced to low standard redox potential of H2O2/O2 (0.695V), resulting in the tendency of H2O2 decomposition.

Prussian blue (PB) nanozymes, one of the promising iron-based nanozymes, are alternately connected by Fe2+-C≡N-Fe3+ to form cyanide-bridged cubic networks. It is worth mentioning that, unlike the catalytic mechanism by which IONPs generate free radicals, the tunable energy levels and abundant redox potentials of PB nanozymes confer them as electron transporters to mimic multienzyme activities (e.g., POD, CAT, SOD, GPx, ascorbate oxidase and ascorbate peroxidase).70

Typically, PB nanozymes can be oxidized into Berlin green (BG) or Prussian yellow (PY) and reduced into Prussian white (PW). (Figure 8a) At lower pH, high active H2O2 oxidize PB to BG/PY (1.4V), which transfer electrons from TMB to H2O2, acting as POD-like enzymes. At higher pH, PB mainly exhibits CAT-like activity, and the reaction processes are as described by Figure 8b. What is more, the redox potentials of O2/O2- and O2-/H2O2 are 0.73V and 1.5V, respectively, and the SOD activity can also be simulated by the processes shown in Figure 8b.70



Figure 8. (a) Mechanism diagram of the multienzyme-like activities of PB nanozymes based on the different standard redox potentials. (b) Reaction equations of POD-, CAT- and SOD-like activities of PB nanozymes. (Reprinted with permission from ref 70. Copyright 2016 American Chemical Society.)

Recently, Dong et al. demonstrated that the POD-like activity of PB nanozymes principally stems from FeNx units rather than FeC6 units in their cubic networks.71 The intrinsic heme-like structure might be conducive to the formation of high active Fe(Ⅳ)=O intermediate.

3.1.2

Cobalt oxide nanozymes

Due to the high redox potential of Co3+/Co2+ (1.3 V), cobalt oxide nanozymes (Co3O4) also possess pH-dependent multienzyme-like activities and the catalytic mechanism is similar with PB nanozymes.72 (Figure 9)

Wang et al. reported for the first time that the cubic Co3O4 exhibit intrinsic POD- and CAT-like activities.73 With methods of cyclic voltammetry and amperometric


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responses, the mechanism of POD-like catalytic activity was established through electrocatalytic behavior of Co3O4 modified glassy carbon electrode about the reduction current of H2O2. The reduction current increased dramatically when H2O2 added. This finding provided a strong support that Co3O4 could transfer electrons from electron donor (e.g. electrode or TMB) to electron acceptor (e.g. H2O2). Therefore, they believed that TMB adsorbed on the surface of Co3O4 provided lone pair electrons from amino group to Co3O4. After accepting electrons, the electrons density and mobility in Co3O4 NPs increased, which accelerated the electrons transfer from Co3O4 NPs to H2O2.73 This conclusion is consistent with the research results of Zhang and Gu et al.72 Moreover, Zhang et al. also confirmed that Co3O4 also had CAT- and SOD-like activities under neutral/alkaline conditions. Since the high redox potential of Co3+/Co2+, the oxidation capacity of Co3O4 was very strong and H2O2 was easily oxidized by Co3+, exhibiting much stronger CAT-like activity than iron oxide nanozymes.72

Figure 9. Mechanism diagram of the multienzyme-like activities of Co3O4 nanozymes

3.1.3 Cerium oxide nanozymes Owing to the fact that the coexistence of Ce3+, Ce4+ and compensating oxygen vacancies, cerium oxide nanozymes (nanoceria) exert remarkable antioxidation and catalytic



properties.74 In the past decade, the mechanism behind their antioxidation has been explored and preliminary conclusions have been obtained.

Flip-flop between the two oxidant states (Ce3+/Ce4+) allows nanoceria to react with superoxide or H2O2, mimicking two important antioxidant enzymes, SOD and CAT. Higher ratio of Ce3+/Ce4+ correlates with higher SOD activity. Whereas nanoceria with lower Ce3+/Ce4+ is more prone to exhibiting CAT activity.75-77 In addition, methyl violet experiments showed that nanoceria could also scavenge OH.78 And this capacity inversely correlates with the size of particles. The smaller nanoceria, the more Ce3+ and oxygen vacancies on its surface, resulting in stronger OH scavenging capacity.

Recently, Yan et al. clarified the antioxidant mechanism from the changes of valence state and coordination structure of Ce before and after adding peroxide species.79 When H2O2 was added, the UV-vis absorption spectra of nanoceria appeared red-shifted and X-ray absorption fine structure (XAFS) measurements indicated that the adsorbed peroxide species slightly increased the coordination number of Ce in the first coordination layer of nanoceria. As depicted in Figure 10, coordinated peroxide species underwent an interfacial redox reaction with nanoceria, leading to the generation of oxygen vacancies (Vo) and the decrease of Ce coordination number. Whereafter, with the oxidation of Ce3+, Vo was released. In general, the redox ability of the peroxide species-coordinated Ce sites plays an important role in the antioxidant performance of nanoceria.79


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Figure 10. Proposed antioxidation process of nanoceria with H2O2. (Reprinted with permission from ref 79. Copyright 2015 Royal Society of Chemistry.)

3.1.4

Single-atom nanozymes

Single-atom catalysts (SACs) have attracted widespread attention in the field of heterogeneous catalysis due to their high metal atomic utilization, excellent selectivity and catalytic activity. Recently, by virtue of their atomically dispersed metal active centers, SACs have become a new candidate in nanozymes field, known as single-atom nanozymes (SAzymes).

Liu, Yan and Fan et al. fabricated the zeolitic-imidazolate-framework (ZIF-8)-derived carbon nanomaterials with a zinc-centered porphyrin mimic structure (PMCS) via a pyrolysis strategy under the protection of mesoporous silica.80 PMCS could serve as SAzymes and possess the excellent POD-like activity. Using a series of ZIF-8 derived nanomaterials obtained at different pyrolysis temperatures as comparison, the structural evolution of ZIF-8 during high temperature was studied. It was found that the PMCS obtained at 800 °C had the most atomically dispersed zinc atoms which were confirmed to be the sources of high catalytic activity. Through the calculation of DFT, it was revealed that the coordinatively unsaturated Zn-N4 structure in PMCS was the active sites. The possible catalytic mechanism was shown in Figure 11. (i) H2O2 was adsorbed on the Zn-N4 active site. (ii) The activated H2O2 generated two OH* via homolysis. (iii) One of the OH* desorbed from the zinc site to produce an active OH. (iv) After the catalytic reaction with other substrates, Zn-OH turned back to its initial state.80



Figure 11. The proposed POD-like catalytic mechanism of PMCS. (Pale blue: Zn, blue: N, gray: C, red: O, white: H) (Reprinted with permission from ref 80. Copyright 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3.2 Noble metal nanozymes Some noble metal nanozymes (e.g., Au, Ag, Pt, Pd, Ir) have also been found to possess enzymatic-like activity.2, 18, 21, 81-92 Interestingly, the catalytic mechanism of them is different from metal compounds-based nanozymes. It is related to the adsorption, activation and electron transfer of substrates on metal surfaces rather than the changes of metal valence.

Noteworthily, these metal nanozymes possess intrinsic pH-switchable POD- and CATlike activities.93 Catalytic mechanism studies have shown that the adsorption of H2O2 on metal surface is the first step to initiate the catalytic reaction. According to the lowest energy barrier principle, the adsorbed H2O2 (H2O2*, * means species adsorbed on metal surfaces) undergoes two different decomposition pathways depending on the pH conditions: acid-like decomposition and base-like decomposition.93


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As an example, Figure 12a apparently shows the calculated reaction energy profiles for the two pathways on the Au {111} surface. Namely, under acidic conditions, preadsorbed H on the metal surface exerts little influence on the decomposition of H2O2 through the base-like pathway. The O-O bond of H2O2* is broken first, producing two OH*. Subsequent reactions result in the formation of H2O* and O*. O* is highly oxidizing and readily extracts H atoms from the organic substrates to render the metal exhibit POD-like activity.93 However, under base conditions, pre-adsorbed OH on the metal surface makes H2O2 more prone to acid-like decomposition, which makes the metal exhibit CAT-like activity. H2O2* firstly destroys the O-H bond, resulting in H* and HO2*. The decomposed H* combines with the pre-adsorbed OH* to yield H2O*, while HO2* passes H to another H2O2*, remaining O2* and transforming H2O2* to H2O* and OH*.93 The mechanism schematic diagram of the pH-switchable POD- and CAT-like activity for metal nanozymes is shown in Figure 12b.



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Figure 12. (a) The calculated reaction energy profiles for two decomposition pathways of H2O2 adsorbed on Au {111} surface. (Inset) Reaction equations of pH-switchable POD- and CAT-like activities of Au nanozymes. (b) The mechanism schematic diagram of the pH-switchable POD- and CAT-like activity for metal nanozymes. (Recomposed with permission from ref 93. Copyright 2015 Elsevier.)

The catalytic mechanism for OXD-like activity of these noble metal nanozymes can be well explained by the dissociative adsorption of O2, which serves as a universal method to activate molecular oxygen.94 It is well known that ground state oxygen is triplet oxygen

and

direct

reaction

with

organic

compounds

is

kinetically

and

thermodynamically disfavored.21 O2 needs to dissociate into single-atomic O-adatoms on metal and its antibonding π* orbitals accept spin-down electrons out of the metals. On this account, the bond order of O2 is decreased to zero. The hydrogens from surrounding organic substrates, such as TMB and ascorbic acid, can be abstracted


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through these O-adatoms. In the whole reactions, metals serve as OXD-like (Equation 4 and 5).94 𝑂2 = 2𝑂 ∗

(4)

𝑂 ∗ + S→𝐻2𝑂 ∗ + 𝑆𝑜𝑥

(5)

Using density functional theory (DFT), Gao et al. calculated the adsorption energies (Eads’s), activation energy barriers of the dissociation (Eact’s) and corresponding reaction energies (Er’s) between dissociation and adsorption states of adsorbed oxygen (3O2 and 1O2) on facets Au (111), Ag (111), Pt (111) and Pd (111) (Figure 13).94 According to the calculation results, presumably, stronger adsorption between oxygen and metal, lesser Eact’s and largely negative Er’s were more favorable for the OXD-like activity of these metal nanozymes. As a proof of concept, the catalytic activities of these metals were investigated by the oxidation of sodium ascorbate and showed decrease in the order of Pd > Pt >> Ag and Au, which was consistent with the results calculated by DFT.94



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Figure 13. Two-dimensional potential energy surfaces for the adsorption and dissociation of O2 on facets of (a) Au (111), (b) Ag (111), (c) Pt (111) and (d) Pd (111). (Reprinted with permission from ref 94. Copyright 2015 American Chemical Society.)

O2- is Bronsted base and easily captures protons from H2O to form HO2. The adsorption of HO2 on metal surfaces is highly exothermic and facile. Once adsorbed on metal, HO2* can easily rearrange and convert to O2* and H2O2*, making the metal exhibit SOD-like activity (Equation 6 and 7).94 𝑂2 ∙

―

+ 𝐻2𝑂 = 𝐻𝑂2 ∙ +𝑂𝐻 ―

2𝐻𝑂2 ∙∗= 𝑂2 ∗ + 𝐻 2 𝑂 2 ∗

(6) (7)

Similarly, some alloy-metallic nanozymes such as Au@M (M = Pd, Pt, Ag), Au@PtAg have also been reported to possess enzyme-like activity.93-97 Gao et al. have demonstrated that the catalytic mechanisms mentioned above were equally applicable to these alloy nanozymes.93, 94

3.3 Carbon-based nanozymes Among the non-metallic nanozymes, the enzymatic activity of carbon-based nanomaterials has been attracting great attention, such as fullerenes and their derivatives, graphene oxide, carbon nanotubes, carbon nanodots. There have been many reports demonstrating their superior ability to mimic the catalytic activity of POD, CAT or SOD.98-105 However, the research work about their catalytic mechanism is relatively rare.

Qu et al. investigated the catalytic mechanism of POD-like activity for graphene quantum dot (GQD) by selectively inactivating the ketone carbonyl, carboxyl or hydroxyl groups.101 The results showed that the -C=O groups could act as the catalytically active sites and the -O-C=O- groups were the substrate-binding sites. Whereas, -C-OH groups were capable of inhibiting activity. Gao and Zhao et al.


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explored the mechanism of POD-like for nanocarbon oxides at the molecular level.104 They revealed a radical mechanism and considered the carboxyl groups to be the reactive sites. What is more, the aromatic domains linked to carboxyl groups were also important for POD-like activity.

More detailed knowledge about carbon-based nanozymes can be acquired from some good research work and reviews.100, 101, 104, 106

4. APPLICATIONS There have been many reviews detailing the applications of nanozymes in biomedical research.107-110 Due to space limitation, only a few typical and well-designed cases are introduced herein to demonstrate the unique and irreplaceable advantages of nanozymes.

4.1 Bioassay As an alternative to natural enzymes, the most widely used of nanozymes is bioassays that can be divided into in vitro and in vivo detection. Since IONPs were first introduced in immunoassay to replace horseradish peroxidase (HRP) in 2007, a large number of subjects were detected with nanomaterials, ranging from metal ions (e.g., Hg2+, Cu2+, Ag+), bioactive molecules (e.g., H2O2, glucose, choline, lactic acid, dopamine, glutathione) to amino acids, nucleic acids, protein markers and cells.23-25, 84, 111-118

Shown as Figure 14, many detection methods have been developed and could be roughly categorized into two strategies: (1) nanozymes, as signal unit, catalyze the reaction of chromogenic substrates (e.g., TMB, ABTS) with targets or their oxidation products. The gradation of color from chromogenic substrates is directly proportional to the level of detected targets; (2) The catalytic efficiency of nanozymes can be enhanced or inhibited by targets, which can be reflected indirectly through the changes of chromogen coloration or the residue of reactants.



Figure 14. The strategies and methods of bioassays based on nanozymes

H2O2 and glucose are the most common bioactive small molecules to be detected.84, 111, 118

For example, Pt-MoO3 hybrid nanomaterials exhibited synergistically enhanced

POD-like activity towards the oxidation of TMB in presence of H2O2.118 Combined with glucose oxidase (GOx), glucose could be specifically detected as low as 187.4 nM with a linear range of 5-500 μM. (Figure 15) In addition, the test result could also be recognized by naked eye. Moreover, the detection of other biomolecules such as lactic acid, dopamine and glutathione, have also been widely reported using the enzymatic properties of nanomaterials or in combination with their natural oxidases.


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Figure 15. (a) The colorimetric detection of glucose based on GOx and Pt-MoO3 hybrid nanomaterials. (GA means gluconic acidox and oxTMB means oxidized TMB.) (b) Calibration plot for glucose detection. (c) The selectivity of this method for glucose. (Reprinted with permission from ref 118. Copyright 2014 Royal Society of Chemistry.)

Compared to double-stranded (ds)- DNA, single-stranded (ss-) DNA is easier to noncovalently bind with AuNPs, blocking the OXD-like activity of AuNPs. DNA hybridization has been demonstrated to specifically switch on their catalytic activity.113, 119

Based on this idea, Fan et al. designed a catalysis-based nanoplasmonic Au probe

for biomolecular detections (e.g., DNA, micro RNA and K+).113 The OXD-like activity of AuNPs, as well as its coupled POD activity of HRP, formed a cascade of glucose oxidation to amplify the noncovalent interaction of DNA with AuNPs, which provided efficient quantitative detection for target DNA. (Figure 16, path a) ABTS-based colorimetric and chemiluminescent detection acquired 14 nM and 0.75 nM detection limit, respectively. Moreover, this strategy was also suitable for aptamer-based assays to detect any small molecules, proteins or cells that have specific aptamers. In addition, the catalytic AuNPs were also associated with AuNP-mediated seed growth. When



adding glucose and HAuCl4, the H2O2 generated in situ could reduce HAuCl4 into Au0 and deposit on the surface of AuNP seeds, gradually increasing the size of AuNPs. Similarly, hybrid DNA could effectively control the quantity of H2O2 by tuning the OXD-like activity of AuNPs, thereby modulating the growth of AuNP-seeded. (Figure 16, path b) The size of AuNPs observably influences their localized surface plasmon resonance (SPR) properties, which is useful for developing highly sensitive bioassays and cell/tissue imaging.113

Figure 16. The regulation of DNA hybridization on the OXD-like activity of AuNPs. (path a) This regulating effect can be amplified via the POD activity or chemiluminescence variations of coupled HRP. (path b) DNA hybridization can increase the size of AuNPs during seed-mediated growth. (Orange strand: target; green strand: adsorption probe) (Reprinted with permission from ref 113. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

When combined with specific antibodies, nanozymes can also act as targeting probes to detect tumor marker proteins. For example, epidermal growth factor receptor (EGFR), a transmembrane glycoprotein overexpressed in many solid tumors, is closely associated with tumor proliferation, metastasis and apoptosis inhibition.120, 121 Zhang and Gu et al. synthesized dimercaptosuccinic acid (DMSA) modified Co3O4 nanopolyhedrons (Co3O4 NHs) via co-precipitation method.114 Co3O4 NHs had excellent POD-like activity and could catalyze the oxidation of diaminobenzidine (DAB) by H2O2 to produce brown color. Nanoprobe targeting EGFR of non-small cell lung cancer (NSCLC) tissues were obtained by coupling the surface-adsorbed Co2+ with the His-Tags at the C-terminal of EGFR single-domain antibodies (EGFR sdAbs). Incubating with NSCLC, this nanoprobe showed outstanding sensitivity and specificity


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towards EGFR through DAB chromogenic reaction. (Figure 17)

Figure 17. (a) Illustration of Co3O4 nanoprobes. (b) The immunohistochemistry detection of EGFR of NSCLC tissues using the POD-like activity of Co3O4 nanoprobes. (Reprinted with permission from ref 114. Copyright 2017 Elsevier.)

Except in vitro assays described above, nanozymes also have great promise for in vivo detection. Wei et al. designed a POD-mimicking nanozyme via in situ growing AuNPs into metal-organic framework MIL-101.115 The obtained AuNPs@MIL-101 was assembled with GOx or lactate oxidase (LOx) to form integrated nanozyme that could oxidize substrate (glucose or lactate) to produce H2O2. The Raman-inactive reporters leucomalachite green were converted into active malachite green (MG) in presence of H2O2. In situ generated MG could be used for SERS detection. At the same time, AuNPs as SERS substrates could enhance the Raman signal of MG. Especially, the changes of glucose or lactate related to physiological and pathological conditions in living brains were easily monitored by this integrated nanozyme. (Figure 18) What is more, the drugs therapeutic efficacy for alleviating cerebral ischemic injuries could also be evaluated via this SERS bioassays.115



Figure 18. Schematic illustration of glucose or lactate detection via the cascade reactions of AuNPs@MIL-101@oxidases in vivo. (Reprinted with permission from ref 115. Copyright 2017 American Chemical Society.)

4.2 Imaging Early diagnosis has always been the bottleneck and key to conquering cancer. Tumor visualization is essential to solve this problem. Although there are many imaging methods such as magnetic resonance imaging (MRI), computed tomography (CT), ultrasound imaging (US), photoacoustic imaging (PAI) and photothermal imaging (PTI), each of them still has some flaws unresolved, making them difficult to be utilized in their full extent. The advances in nanotechnology motivate the development of nanozyme-based catalytic imaging to overcome the shortcomings of traditional imaging technology.

In pathological state, excessive H2O2 can serve as potential diagnostic marker. For instance, Nie et al. modified graphene quantum dot nanozyme (GQDzyme) with ABTS to construct GQDzyme/ABTS nanozymes which are H2O2-triggered PAI contrast agents.122 Due to the POD-mimicking activity, GQDzyme/ABTS could effectively convert ABTS into its oxidized form with strong near-infrared (NIR) absorbance. Furthermore, inspired by exosomes, they camouflaged GQDzyme/ABTS nanozymes with folate (FA)-linked erythrocyte membranes (RM) to design the exosome-like nanozyme vesicles. It could be speculated that the camouflage effect of RM endowed nanozymes a long circulation time in blood and improved accumulation in tumors. As verification, the nanozyme vesicles were injected intravenously into CNE-2 tumor-


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bearing mice. RM cloaking and FA targeting, as expected, helped the vesicles escaping the entrapment of reticuloendothelial system (RES) and specifically recognized tumor cells, acting as ideal H2O2-responsive PAI contrasts for tumor deep -tissue imaging in vivo.122 (Figure 19)

Figure 19. (a) Schematic illustration of GQDzyme/ABTS nanozymes for H2O2-triggered PAI contrast agents of tumor cells. (b) PAI for nasopharyngeal carcinoma detection with exosome-like nanozyme vesicle in CNE-2 tumor-bearing mice. (c) PA signal intensity of panel b. (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001) (Reprinted with permission from ref 122. Copyright 2018 American Chemical Society.)

Very recently, Multimodal imaging technology has been developed to realize the combination of the respective advantages of each imaging methods and provide more comprehensive and accurate information for disease diagnosis. An ingenious strategy in which PBNPs could be US and MRI dual modality imaging probes for the diagnosis of H2O2 with excellent resolution and sensitivity was proposed by Gu et al.123 Under neutral condition, PBNPs possessed CAT-like activity, which could not only alleviate



oxidative stress in inflammation mice, but also produce nucleated and paramagnetic O2 as both ultrasound and T1 MRI contrast agents for investigation of H2O2 generation.

4.3 Therapeutics 4.3.1 Cytoprotection As early as 1991, Krusic et al. speculated that fullerenes could be used as free radical scavengers, known as "free radical sponges".124 Shortly thereafter, Dugan et al. demonstrated that soluble fullerenes possess the capacity of scavenging O2- and H2O2, and serve as neuroprotective agents to greatly reduce over-activation and apoptosis of cortical cells and to effectively alleviate functional deterioration and death in the mouse model of amyotrophic lateral sclerosis.125, 126 Due to the essential role of nanozymes in antioxidant, an increasing research interest has been focused on their cytoprotection, anti-aging, anti-inflammatory effects. (Table 4) Table. 4 Nanozymes act as antioxidant for neuroprotection and cytoprotection Nanozymes

Enzyme-like

Protection mechanism

Application

Ref

activity Metal compounds-based

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 61

Fe3O4 NPs

CAT

127

·Preventing the decrease of anti-apoptotic

Delaying aging and

protein Bcl-2 and the increase of pro-apoptotic

ameliorating

protein BAX caused by H2O2 in L929 cells

neurodegeneration in

·Reducing the protein levels of α-Synuclein

aged Drosophila

and the activation of Caspase-3 in PC12 cells of Parkinson's disease model CeO2 NPs

CAT, SOD

Protecting the CPCs

128

· Inhibiting the nuclear fragmentation and

Protecting

129

apoptosis induced by 3-Amino-1,2,4-Triazole (3-

cells

AT) in human hepatic cells (WRL-68) due to the

induced

eliminating H2O2 ability of CeNPs (+4)

and preventing hepatic

·Re-establishing the glucose metabolism of 3-

injury

AT treated cells by CeNPs.

oxidative stress

·Reducing the ROS levels of cardiac progenitor cells (CPCs)

CeNPs

CAT

WRL-68

from

3-AT

acatalasemia caused

by

·Altering the expression of mRNA and proteins related to H2O2 metabolism PEGylated CeNPs

SOD

·Decreasing the buthionine sulfoximine (BSO)-

Acting as antioxidant

induced ROS levels in human keratinocytes

and


antigenotoxic

130

Page 39 of 61

(HaCaT cells)

agent to protect HaCaT lactate

cells from oxidative

dehydrogenase (LDH), nuclear fragmentation

stress caused by GSH

and micronucleus formation

depletion

·

Decreasing

the

release

of

· Overseeing the expression of antioxidant proteins PB NPs V2O5 nanowires

POD, CAT,

·Scavenging ROS

Reducing

colitis

SOD

·Inhibiting proinflammatory cytokine

mice

GPx

·Reducing H2O2 in mammalian cells

Cytoprotection

in

131

132

·Restoring cellular redox balance Mn3O4

CAT, SOD,

·Scavenging OH in SHSY-5Y cells of

GPx

Parkinson disease-like model

Rescuing cells from

133

neurotoxin-induced oxidative damage

PAMAM

CAT

dendrimerencapsulated

Noble metal-based

Pd NPs

Protecting mice

can inhibit the formation of ·OH.

primary neuron cells

83

retaining CAT-like activity in physiological

(AuNC-NH2) Pt NPs

·The surface tertiary amines of AuNCs-NH2 ·AuNCs-NH2 lose POD-like activity while still

Au nanoclusters

conditions. POD, CAT,

·Reducing ROS levels in mouse embryonic

Protecting MEF cells

SOD

fibroblast (MEF) cells of human Cerebral

from oxidative stress

Cavernous Malformation (CCM) disease model

damage

·Maintaining the mitochondrial membrane

Protecting human

potential

umbilical vein

·Scavenging H2O2

endothelial cells

CAT, SOD

18

2

·Preventing damage to lipid, DNA and protein caused by oxidative stress Ir NPs

POD, CAT

·Scavenging ROS

Reducing the oxidative

91

damage of A549 lung cancer cells C60-methionine derivate based

Carbon-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


_

·Increasing the GSH levels

Protecting lead-

·Reducing malondialdehyde content

exposed

·Attenuating DNA damage

human neuroblastoma SH-SY5Y cells

4.3.2 Tumor therapy based on ROS regulation Moderate levels of ROS can function as critical second messengers during cell proliferation, pathogen defense and homeostasis. In comparison with normal cells, tumor cells exhibit a relatively high ROS level which stimulates the expression of oncogenes and plays an important role in tumor proliferation, metastasis and


134


neovascularization.135-137 In addition, abnormalities in ROS levels usually lead to genetic instability and even gene mutations that induce tumor cells resistant to many gene-targeted drugs.

In fact, the redox ability of nanozymes can be used to disturb the delicate ROS balance in tumor cells for therapeutic purposes.138 (Figure 20) On the one hand, as described in Section 4.3.1, nanozymes can serve as antioxidants to remove the excessive high-toxic ROS and reduce tumor multidrug resistance, achieving therapeutic effects; on the other hand, pro-oxidative properties of nanozymes can be used to break the ROS threshold of cells, resulting in oxidative damage and then tumor cell apoptotic or necrotic.

Figure 20. The bi-directional regulating effect of nanozymes in intracellular ROS. (Reprinted with permission from ref 138. Copyright 2018 Chinese Academy of Sciences.)

The availability and versatility of this new-style tumor therapy strategy have been successfully demonstrated at both cellular and animal levels. For instance, Shi et al. integrated natural GOx and ultra-small Fe3O4 nanozymes into mesoporous dendritic silica NPs to fabricate the biodegradable and biocompatible nanocatalysts (GFD NCs).62 GFD NCs could enter tumor tissues through enhanced permeability and retention (EPR) effect when administrated intravenously into 4T1 mammary tumor and U87 glioblastoma xenograft mice. Under mild acidic tumor microenvironment,


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glucoses were gradually depleted and a considerable amount of H2O2 was produced for following Fenton-like reaction catalyzed by Fe3O4 nanozymes, resulting in the generation of highly toxic OH that can trigger the apoptosis and death of tumor cells. (Figure 21)

Figure 21. Schematic illustration of GFD NCs for sequential catalytic-therapeutic. (Reprinted with permission from ref 62. Copyright 2017 Nature Publishing Group.)

Similarly, Gao et al. demonstrated that N-doped carbon nanozymes (N-PCNSs) possess multienzyme-like activities (OXD, POD, CAT and SOD) which were responsible for the regulation of ROS in vivo.61 Ferritin was introduced as mediator to specifically guide N-PCNSs into lysosomes via receptor-mediated endocytosis. In lysosomes, O2 and H2O2 were converted into free radicals via the OXD/POD-like activities of NPCNSs accompanied with the consumption of O2. (Figure 22a-d) In vivo experiments had demonstrated that N-PCNSs were capable of specifically targeting HepG2 and HT29 cells, reducing tumor volume and improving survival rate of tumor-bearing mice. (Figure 22e, f)



Figure 22. (a) Schematic of tumor cell destruction based on H-ferritin nanoparticles (HFn)coordinated N-PCNSs. (b) TEM image of HFn-N-PCNSs. Scale bar: 50 nm. (c) TEM image of HFn-N-PCNSs located in the lysosome of tumor cell. Scale bar: 500 nm. Red arrows: the position of HFn-N-PCNSs. (d) Quantitative analysis of HFn-enhanced N-PCNS internalization by flow cytometry. (n = 3, ****p < 0.0001) (e) Photograph of tumors treated with HFn-N-PCNSs. (n = 5) (f) The survival percent of tumor therapy with HFn-N-PCNSs. (Reprinted with permission from ref 61. Copyright 2018 Nature Publishing Group.)

Owing to the tumor hypoxia microenvironment, the therapeutic efficiency of ROSmediated photodynamic therapy (PDT) or chemodynamic therapy (CDT) is greatly impaired. For instance, PDT is one of the widely applied methods for ROS-mediated cancer therapy using photodynamic effects between photosensitizers, light and oxygen.139, 140 However, there still remain several unresolved problems in traditional PDT, such as dependence on oxygen, limited light penetration depth, and systemic toxicity caused by photosensitizer autocatalysis and off-site localization, which make it not a first-line treatment option.

Fortunately, a large number of CAT-mimicking nanozymes can decompose tumor-rich H2O2 into O2 to alleviate hypoxic microenvironment, facilitate the generation of 1O2


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and aggravate the damage of cancer cells. Moreover, some nanomaterials, such as copper ferrite nanospheres (CFNs) and porphyrin modified Fe3O4 nanoparticles, possess enhanced POD-like activity to convert the endogenous H2O2 to O2 and OH through the light-enhanced Haber-Weiss reaction (Equation 8), which not only alleviate hypoxic environment, but also increase the cancer-killing effects.141, 142 𝑂2― · + 𝐻2𝑂2 𝐻 + 𝑂2 + 𝐻2𝑂 + ·𝑂𝐻

(8)

Chen and Nie et al. constructed an activatable 1O2 generation system by grafting IONPs with linoleic acid hydroperoxide (LAHP).143 Once IO-LAHP NPs entranced tumor cells through endocytosis, oxygen-containing LAHP molecules were decomposed into 1O2 through the Russell mechanism in the presence of released Fe2+ from IONPs.144, 145 (Figure 23a) Compared with control group, the cells treated with IO-LAHP NPs revealed obviously membrane disruption, cytoplasmic vacuolation, chromatin condensation and fragmentation and the presence of apoptotic bodies. (Figure 23c, d) Moreover, the results of U87MG tumor-bearing mice experiment also showed that IOLAHP NPs could efficiently inhibit tumor growth.143 (Figure 23b, e) This tumorspecific 1O2 production strategy is independent of O2 or other additional stimuli, which effectively overcomes the disadvantages of PDT.



Figure 23. (a)Schematic of cancer therapy via activatable 1O2 generation system. (b) MRI and (c) hematoxylin and eosin (H&E) staining of mouse tumors treated with IO-LAHP and IO-LA (control) NPs. Yellow arrows: the potential lesion in tumors. (d) TEM images of a healthy (control) and a tumor treated with IO-LAHP NPs. Red dotted square: the chromatin condensation and fragmentation. Blue dotted square: apoptotic bodies. (e) Tumor growth curves of mouse treated with different formulations. Black triangles: three doses every three days. (n = 5, **p < 0.01) (Reprinted with permission from ref 143. Copyright 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

4.3.3 Combination therapy Very recently, the merging of imaging and therapy technology has ignited extensive research interests for constructing integrated theranostic agents. Noteworthily, in addition to catalysis, nanomaterials endow nanozymes with more unique nanoscale properties including optical, electrics, magnetics and thermal performances, which make them more powerful than natural enzymes. These nanoreagents play an irreplaceable role in tumor combined treatment. Some examples are summarized in Table 5.


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Table. 5 The applications of integrated theranostic nanoreagents in tumor combined treatment Nanoreagents

Role of enzyme-like

Imaging

Therapeutics

Ref

Copper ferrite

Fenton and Fenton-

·MRI: CFNs possess ultrahigh

·PDT: CFNs can catalyze H2O2 into

nanospheres (CFNs)

like reaction

traverse relaxivity (468.06 Mm-1s-

O2 to relieve tumor hypoxia.

1)

·CDT: Fenton reactions can occur to

and can be used as T2 contrast

agents.

141

produce OH under 650 nm laser due to the presence of two redox pairs (Fe2+/Fe3+ and Cu+/Cu2+) in CFNs. ·Photothermal therapy (PTT): CFNs have high photothermal conversion (PTC) efficiency under irradiation of 808 nm laser.

Bovine serum

CAT-like:

albumin-iridium oxide

protecting

nanoparticles (BSA-

cells

normal

IrO2 NPs)

146

·CT: BSA-IrO2 NPs have high

·Photocatalysis-induced PDT

X-ray absorption coefficient.

·PTT: extraordinary PTC efficiency

·O2 bubble-enhanced PAI ·PTI: extraordinary PTC efficiency

Manganese ferrite

Fenton reaction

nanoparticle-anchored

147

· MRI: MFMSNs exhibit T2

·PDT: MFMSNs can work as

contrast effect.

Fenton catalyst to produce sufficient

mesoporous silica

O2 for PDT in H2O2-rich humor

nanoparticles

microenvironment.

(MFMSNs) Chitosan-encapsulated Fe3O4 NPs modified with CuS and porphyrin (FCCP NPs)

Ultrasmall Cu2ZnSnS4 (CZTS) nanocrystals

POD-like:

·PAI: CuS serves as PAI agents.

·FCCP NPs act as CAT to provide

producing ROS from

·Photoluminescence imaging.

O2 for oxygen-dependent PDT.

endogenous H2O2

(PLI): Porphyrin has PL property.

Porphyrin acts as photosensitizer.

CAT-like:

·MRI: FCCP NPs possess high

·PTT: CuS serves as PTC agent.

generating O2 to

r2 relaxivity for T2 imaging.

overcome cellular

·PTI: FCCP NPs possess good

hypoxia

PTC efficiency.

POD-like:

·PAI: CZTS possess high NIR

PTT:

decomposing H2O2

absorption.

absorption and PTC efficiency.

into ROS due to the

·MRI: The improved T1 imaging

presence of Cu+

is due to the enhanced longitudinal relaxivity caused by CZTS.


CZTS

possess

high

NIR

142

148


5. CONCLUSION AND CHALLENGES Nanozymology, an emerging interdisciplinary field, is full of limitless vitality and challenges. Compared with natural enzymes, nanozymes have four prominent characteristics (Figure 24): (i) Designability: The catalytic performance of nanozymes is affected by many factors such as size, shape, modification and composition. According to practical application, various methods can be used to regulate and tailor nanozymes toward high catalytic efficiencies; (ii) Maneuverability: Nanomaterials possess unique nanoscale properties, such as fluorescence, electricity, magnetism, photothermal and photoacoustic conversion capabilities, which endow nanozymes excellent controllability; (iii) Multifunctionality: The ingenious combination of enzyme-mimicking activities of nanozymes and their unique physicochemical properties or biological properties (such as EPR effect for tumor) is a common strategy for constructing the multifunctional integrated theranostic nanoplatform, making nanozymes versatile in biomedical field; (iv) Applicability: Some nanozymes have been proved to possess druggability, such as IONPs act as iron-supplementary and PBNPs serve as the antidote of thallium (Tl) elements. Furthermore, these nanomaterials-based artificial enzymes take the advantages of improved stability, low cost in preparation and excellent tunability in catalytic, which lay a good foundation for the potential application of nanozymes.


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Figure 24. Schematic diagram of nanozymes’ characteristics

Although many advances in this young field, some issues still remain elusive and need further investigation.

(i) Nanozymes, the mimics of natural enzymes, are not always comparable to natural enzymes in some respects. The pursuit of higher catalytic activity and good substrate specificity has been the direction of researchers' efforts. In addition, most of the catalytic types of nanozymes are currently limited to redox reaction. With the continuous advancement of synthetic technology, nanozymes based on novel materials and catalytic types may be developed to meet the demand of biomedical applications.



(ii) From the perspective of bionics, it is well known that natural enzymes have their lifecycle and are expressed by gene in a timely manner. That is to say, under normal physiological conditions, they can be expressed as needed and degraded automatically. It is still unclear whether nanozymes can simulate this intelligence to truly replace natural enzymes in the future. There is still a long way to go for nanozymes to learn from natural enzymes.

(iii) At present, knowledge about the underlying catalytic mechanism of nanozymes is limited and it is imperative to decode it in an effort to acquire superb catalytic activity. More advanced characterization methods and multidisciplinary expertise are needed to unveil those general rules. For instance, the recently developed SAzymes with atomically dispersed metal active centers can mimic the structure of natural enzymes, providing a breakthrough in exploring the catalytic mechanisms of nanozymes at the atomic level.

(iv) The catalytic activity of nanozymes is determined by the intrinsic properties (e.g., size, shape, composition) and the extrinsic factors (e.g., surface modification, pH, temperature, reaction medium). When translating nanozymes into clinical applications, it is difficult to evaluate the complex relationship between their catalytic activity, biosecurity and therapeutic effects.149 In certain cases, the multi-enzymatic activities of nanomaterials may cause some potential side effects in the ROS-mediated diseases treatment to hinder the therapeutic effect, which should be carefully designed.92, 150

(v) When different methods are used to detect the enzyme-like activity of the same nanomaterial, sometimes, the results obtained are quite disparate, which brings an enormous challenge for the fundamental research and the industrialization of nanozymes. Therefore, the establishment of standards related to nanozymes is essential for the long-term development of nanozymology. It is heartening that the first National Standard Method in the field of nanozymes has been reviewed and approved in 2019,


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established by researchers from Southeast University, Institute of Biophysics, Chinese Academy of Sciences, Nanjing Dongna Biotech co., Ltd and Institute of Basic Medicine, Chinese Academy of Medical Sciences. In the future, it is hoped that more relevant standardized methods can be established to unify the measurement of enzyme-like activity of nanozymes and some reference materials can be produced to provide value comparison standard for various nanozymes.

In general, we hope this review will give readers a more comprehensive understanding of nanozymes. And along with unceasing exploration, nanozymes, as next-generation artificial enzymes, are expected to facilitate the development of nanomedicine and have a promising prospect to benefit human health.



Table of Contents Graphic


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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China [No. 2017YFA0205502]; National Natural Science Foundation of China [No. 61821002, 81571806, 81671820]; the Science and Technology Support Project of Jiangsu Province [No. BE2017763]; and the Fundamental Research Funds for the Central Universities.

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