The rational design of nanozymes for imaging-monitored cancer therapy

Zhe Dong , Peng Liang , Youjuan Wang , Guoqiang Guan , Lili Teng , Renye Yue , Chang Lu , Shuangyan Huan , Xia Yin and Guosheng Song *
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China. E-mail: songguosheng12@sina.com

First published on 16th May 2023

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Introduction

Nanozymes are neotype artificial enzymes that are based on nanotechnology and nanomaterials with intrinsic enzymatic properties and catalytic activities and have attracted enormous attention in biological and biomedical applications.^1–9 Since the peroxidase-like properties of iron oxide were discovered in 2007 by Yan,¹⁰ plenty of nanomaterials have been developed to imitate natural enzymes owing to the advantages of nanomaterials, such as increased and customizable catalytic performance as compared to natural enzymes, low cost, convenient, and massive production, as well as high stability.^11–14 Different kinds of nanozymes have been developed and their catalysing abilities have been investigated, including noble metal-based nanomaterials, iron oxide, MoOx, nitrogen-doped porous carbon nanospheres.^15–18 These nanomaterials have been employed to mimic oxidase,^19–21 peroxidase (POD),^22–24 catalase (CAT),^24–26 and superoxide dismutase (SOD)^24,27 to produce cytotoxic reactive oxygen species (ROS) or supplement O₂ by decomposing endogenous H₂O₂. Strategies were reported by utilizing ROS produced by nanozymes to disturb the vulnerable homeostasis of the tumor microenvironment and reverse the tumor hypoxia to enhance treatment effects, offering new opportunities to develop cancer-specific therapeutics.

The therapeutic effects of nanozymes greatly rely on their catalytic activities. Their high catalytic activity would lead to a higher therapeutic effect. However, there are huge differences in the catalytic activities of nanozymes between in vivo and in vitro. The enzymatic abilities and the catalytic properties of nanozymes could be influenced by and interfere with the complicated surrounding biological environment in tumors. Furthermore, the high performance of nanozymes brings out not only comprehensive treatment effects but also increasing side effects to normal tissues. Thus, to evaluate the accuracy of treatment with nanozymes, to report the real-time catalytic activities, and to monitor the therapeutic results, appropriate imaging tools are necessary for monitoring the use of nanozymes in real-time, enabling the prediction of intravital therapeutic effects in vivo and providing guidelines for further dosage. Nevertheless, the existing imaging methods are unsuitable for detecting the activities of nanozymes in vivo. Currently, extra probes are used mostly as indicators to evaluate the catalytic activities of nanozymes through the detection of the corresponding markers in solution or at the cellular level.^28–31 Unfortunately, the in vitro tests were unable to reveal whether a nanozyme would function properly in the extremely complicated biological milieu in vivo. Thus, monitoring the catalysing ability of nanozymes is still a challenge in bioapplications. To date, several fresh imaging methods have been developed and investigated, including chemiluminescence (CL), near-infrared fluorescence (NIRF), photoacoustic imaging (PAI), and magnetic resonance imaging (MRI), and various strategies have been applied in therapeutic and real-time monitoring based on nanozymes and relevant imaging.

In this review, we cover the development and biomedical applications of nanozymes for the treatment of malignant tumors. The current progress in real-time imaging and monitoring of their catalytic activities is discussed. Various appropriate instances are given, including measuring ROS production using optical imaging, imaging biochemical incidents with photoacoustic imaging, and monitoring the ion release of nanozymes by using MR imaging. This review aims to provide some new perspectives to researchers looking for the most recent developments in the imaging monitoring of nanozymes.

Development progress of nanozymes in cancer therapy

The intrinsic limitations of natural enzymes have emerged in the development of artificial enzyme mimics. Benefiting from the unique physicochemical characteristics of nanomaterials, nanozymes can be obtained through rational design for biomedical applications.

Nanozymes have attracted much attention as the next generation of medication in the past 15 years since the advent of “nanozymology”, a new field connected to nanotechnology and biology has emerged around nanozymes. Plenty of studies that investigate the systematic design of nanozymes, catalysis modulation, catalytic mechanism, and monitoring of catalysts have been published. Nanomaterials including iron oxide, carbon nanoparticles, Prussian blue, metal–organic frameworks (MOFs), and alloys (such as FePt), etc., were synthesized and further investigated to lucubrate the therapies with singular or multiple enzyme mimetic activities. For instance, Prussian blue nanoparticles simultaneously exhibited peroxidase, catalase, and superoxide dismutase-like activity;^32,33 Fe₃O₄ nanoparticles displayed pH-dependent peroxidase and catalase-like activities,^34,35 while Mn₃O₄ nanoparticles activated three different types of cellular antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase.^36,37 Using the enzyme-like qualities and physiochemical characteristics of nanoparticles, nanozymes have shown the potential to treat tumors through the catalysis of ROS production (POD, OXD, GOx, etc.) or the relief of tumorous hypoxia (CAT, SOD, etc.). Examples from our group and other researchers are listed and discussed in detail in the following chapters, and perspectives are also provided.

The construction and engineering principles of current nanozymes

To date, various nanomaterials have been developed as nanozymes to treat malignant tumors, including iron oxide, manganese oxide, noble metal-based nanoparticles, etc.³⁸ Although the activities of most nanomaterials are superior to the natural enzymes mimicked, their specificity is still low, which limits their practical applications. More efforts have been expended to enhance the performance of nanozymes, making them superior substitutes for natural enzymes. Nanomaterials play two roles as enzyme mimics in therapeutics. The first is the generation of ROS through POD-like or OXD-like enzyme activity that can directly kill tumor cells by mediating DNA synthesis, protein damage, and so on.³⁹ The second is the release of O₂, which mimics the CAT or SOD enzyme activity that further alleviates tumor hypoxia and promotes tumor cell apoptosis, thus improving the efficacy of other treatment methods^40,41 (Fig. 1).

Herein, we have summarized a series of previous works by classifying them based on the type of material (Table 1). We focused on the methods and strategies to achieve various enzymatic functions with a single nanomaterial and ways to emulate the active core structure of natural enzymes to increase the selectivity and catalytic activity of the enzymes.

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1. Fe-based nanomaterials

In 2007, Yan¹⁰ and coworkers found that Fe₃O₄ magnetic nanoparticles (IONP) exhibited the H₂O₂, pH, and temperature-dependent peroxide-like activity that could catalyze different peroxidase substrates to the typical color change propensity, including TMB, DAB, and OPD. The ferric and ferrous ions available on the surface of IONP endowed Fe₃O₄ with catalysis activity by imitating the active sites within the heme part of horseradish peroxidase (HRP). This finding opened the door to the investigation of nanozymes. As the most common transition metal and the most accessible nanomaterials, IONP and other Fe-based nanomaterials were chosen to simulate natural enzymes in tumor therapies at first, including iron-based MOFs,^42,43 iron oxide,^44–46 Fe–O coordination complexes,⁴⁷ Fe-doped nanoparticles,^48–51 Fe-based polymetallic oxides,^52–54 Fe–N clusters or Fe–N complexes,^55–59etc.⁶⁰

Wang and coworkers utilized the CAT-like and POD-like enzyme activity of IONPs to build synergistic nanotheranostics.⁶¹ Hydroxyl radicals (˙OH) were produced during the POD catalytic process and O₂ was produced in the CAT process that could further boost the phototherapy of IR780. The nanotheranostics exerted an improved anti-cancer effect upon light excitation as a result of the nanozyme catalysis effect-enhanced phototherapy in a triple-negative breast cancer model. Xu presented an engineered Janus structure through the combination of TiO₂ and Fe₃O₄ for augmented sonodynamic (SDT) and chemodynamic (CDT) cancer therapy.⁶² The decomposition of IONPs not only endowed the Janus structure to generate ˙OH but also narrowed the band gap of TiO₂ and reduce the recombination rate of the e⁻/H⁺ pair to accelerate the SDT efficacy (Fig. 2a). Due to the unique magnetic feature of its mesocrystalline structure, the hollow Fe₃O₄ mesocrystal (Fe₃O₄ MCs) that Du and colleagues described displayed high magnetothermal conversion efficiency.⁶³ Because the Fe²⁺/Fe³⁺ ratio was higher and there were many more oxygen defects in the Fe₃O₄ MCs than in Fe₃O₄ NPs, the POD-like activity of the Fe₃O₄ MCs was enhanced. In addition to increasing cell apoptosis to kill cancer cells, ROS created by the Fenton reaction catalyzed by POD-like IONPs nanozymes can also decrease heat shock proteins’ expression. A self-augmented synergistic effect resulted from the acquired ROS, enabling low temperature-mediated magnetic hyperthermia and in situ rising temperature facilitating CDT (Fig. 2b). Several metal-doped Fe-perovskite nanocrystals were created in addition to IONPs for their ability to mimic enzymes. Chang obtained a therapeutic perovskite nanocrystal by using LaFeO₃ with OXD, POD, glutathione peroxidase (GSH-Px), and CAT-like activities.⁶⁴ The cascade catalytic events of LaFeO₃ involved the ongoing production of ROS, reversal of the hypoxic microenvironment, and depletion of endogenous glutathione (Fig. 2c).

Many other Fe-based nanomaterials were also investigated for their biomedical applications as nanozymes. Jiang and coworkers prepared an Fe-doped polydiaminopyridine nanofusiform (Fe-PDAP).⁶⁵ The Fe-PDAP NFs with CAT-like activity overcame tumor hypoxia to enhance the efficiency of photodynamic therapy (PDT) and chemotherapy (CT) (Fig. 2d). Liu developed iron-based Fe–N₅ moieties embedded in N-rich carbon (FeN₅) through a melamine-mediated two-step pyrolysis strategy.⁶⁶ Enzyme kinetic experiments demonstrated that the Fe–N₅ moieties remarkably increased the POD-like activity, and the catalytic efficiency of FeN₅ was higher than those in four-coordinated nanozyme (FeN₄) and Fe₃O₄ NPs. Chang and coworkers developed a nano-platform DCGP (DMSN@CoFe₂O₄/GOD-PCM) with a combination of nanozyme and natural enzyme, which was realized by the deposition of ultrasmall CoFe₂O₄ bimetallic oxide and glucose oxidase (GOD) into the large mesopores of dendritic mesoporous silica (DMSN).⁵² The cascade catalysis reactions of the CoFe₂O₄ nanozymes were TME-activated. GOD strengthened the weak acidity of the tumor area and produced more H₂O₂. CoFe₂O₄ was initiated under a lower pH value to produce toxic ˙OH. Moreover, the photothermal (PTT) effect of CoFe₂O₄ improved the enzyme-like activities.

Song and coworkers also developed several Fe-based nanomaterials for enzyme-mimicking bio-applications.^67,68 Among them, an acidity-unlocked nanoplatform based on FePt nanoparticles (FePt@FeO_X@TAM-PEG) was reported for tumor-specific catalysis and highly efficient anticancer treatment.⁶⁸ The pH-responsive drug tamoxifen (TAM) endowed FePt@FeO_X with tunable catalytic activity in acidic tumor microenvironments. The released TAM within cancer cells could lead to the upregulation of lactate and thereby the accumulation of intracellular H⁺. Through the positive feedback, a massive amount of FePt@FeOx nanozyme was released and was able to produce more ROS by depleting the endogenous H₂O₂ within the more acidic conditions. This self-boosting generation of ROS showed substantial anticancer outcomes in vivo, providing a new choice for tumor-specific cascade catalytic therapy (Fig. 3).

2. Mn-based nanomaterials

Manganese (Mn) is a biologically essential element that is also a crucial cofactor for many metallo-enzymes, including glutamine synthetase (GS), pyruvate carboxylase, and Mn superoxide dismutase (MnSOD). Mn affects the metabolism and redox steady state in tumor cells due to its multiple valences and high spin.^69,70 Many scientists have studied Mn-based nanozymes in the wake of natural selection, and diverse Mn-based nanomaterials have shown a range of enzyme-like functions, including glutathione peroxidase,^3,71–74 catalase,^73,75–80 superoxide dismutase,^80,81 and peroxidase.^82,83

A versatile mesoporous nanozyme was created by Wang and colleagues from MnCo-MOFs to increase the effectiveness of PDT under the guidance of bioimaging⁷⁷ (Fig. 4a). First, a manganese cobalt-based MOF was coated with mesoporous silica to create a mesoporous nanozyme. The created nanozyme was loaded with Ce6 for PDT after the silica shell was removed by annealing and modified with polydopamine and poly(ethylene glycol). The nanozyme could increase tumor hypoxia and boost PDT effectiveness by catalyzing excess H₂O₂. Lv and partners constructed a Mn–CoS nanozyme to mimic CAT, POD, and GSH-Px activities to generate ˙OH and O₂⁸⁴ (Fig. 4b). O₂ could enhance PDT efficacy through the relief of tumor hypoxia. The obtained ˙OH could directly kill tumor cells along with ¹O₂ generated from PDT. Combined with PTT, the Mn-CoS nanozyme showed enhanced nanozyme-mediated phototherapy and excellent anti-tumor ability with trimodal imaging. Zhu synthesized a Mn-based and PEGylated single-atom nanozyme that continuously generated ˙OH and O₂˙⁻ through the parallel catalytic reactions that originated from H₂O₂ and O₂⁸⁵ (Fig. 4c). The coordinated Mn-atoms in ZIF-8 exhibited excellent Fenton-like catalysis activity. Compared with conventional MnO₂, the Mn single-atom nanozyme showed greater enzymatic activity in vitro, disrupting the intracellular redox balance and killing cancer cells more efficiently. Wu synthesized a bimetallic oxide Cu_1.5Mn_1.5O₄ that showed triple enzyme-like activities for bacterial-infected wound therapy⁸⁶ (Fig. 4d). Because of the increased exposure of active edge sites, the obtained Cu_1.5Mn_1.5O₄ displayed elevated triple enzyme-like activities (OXD, POD, and GSH-Px), which might significantly encourage the production of ROS.

Our group developed a series of Mn oxide-based nanomaterials for the enzyme-like catalysis of ROS to treat malignant tumors. In 2019, we reported the MnFeO₄@MOF, which regulated TME via persistent circulating catalysis to enhance PDT.⁷³ Once internalized in the tumor, MnFeO₄@MOFs could continuously produce O₂ to overcome tumor hypoxia via cyclic Fenton reactions. It can persistently consume GSH, decrease ROS depletion during PDT, and achieve a higher therapeutic efficacy in vitro and in vivo. We also designed nanoprobes by mixing imaging methods with Mn-based nanomaterials for the image-guiding of nanozymes for tumor therapies, including MRI,^87,8819F-MRI,⁸⁸ ratiometric fluorescence and photoacoustic imaging.⁸⁹ The specific information on these works is given in the following sections of this minireview.

3. Noble metal-based nanomaterials

Noble metals including Au, Ag, Ru, Rh, Pt, Os, Ir, and Pd were widely used as nanozymes for biomedical applications, especially for tumor therapy.^90–94 Cao and coworkers designed a Cu–Ag alloy to mimic cytochrome c oxidase to change oxygen into the cytotoxic superoxide (O₂˙⁻) and ˙OH by using the tumor-overexpressed cytochrome c to upregulate the oxide stress level and further kill tumor cells.⁹⁵ Li and coworkers synthesized Au/Cu/Fe ternary metal nanosheets to impede the anti-ferroptosis pathway to boost tumor therapy by utilizing nanozyme activities.⁹⁶ In addition to acting as GOx replacements to reduce intracellular glucose, Au NPs also interfered with the pentose phosphate pathway (PPP), inhibiting the synthesis of GSH, and preventing the recycling of coenzyme Q10 (CoQ10). Copper could directly oxidize GSH into GSSG through a reaction. The nanosheets could negatively affect the antioxidant and anti-ferroptotic pathways to increase ferroptosis damage, making them a potent tool for ferroptosis therapy along with the GPX4-deactivating medication RSL3 (Fig. 5a). To achieve hyperthermia-amplified nanozyme catalysis for imaging-guided synergistic cancer therapy, Zhu and colleagues created a Pt-doped Ti₃C₂ nanocomposite.⁹⁷ Under NIR-II laser irradiation with a low power density, PTT can be achieved. The Pt nanozyme with POD-like catalytic activity and Ti₃C₂T_X nanosheets were coupled to synergistically kill cancer cells while reducing side effects and injury to normal tissues from overheating (Fig. 5b).

Sun and coworkers designed and synthesized an ultrasonic switchable nanozyme system by combining Pd/Pt nanosheets with an organic sonosensitizer T790.⁹⁸ The intriguing aspect is that the CAT-like activity of Pd@Pt could be blocked by modifying T790 on the surface of a Pd/Pt nanosheet, and the nanozyme activity was restored following US irradiation. The potential harm and adverse effects of nanozymes in healthy tissues may be reduced as a result of this “block and activate” function. This technology was created to increase the effectiveness of SDT by generating catalytic oxygen and sonosensitizer-mediated ROS in a controlled manner during ultrasound activation (Fig. 5c). To avoid bacterial infections, Zhu claims that a cascaded nanoreactor (Pd-Ru/GOx) was built that could continually convert glucose into the poisonous compound ˙OH through a series of catalytic reactions.⁹⁹ The obtained Pd–Ru/GOx simultaneously displayed GOx activity and POD activity, in which GOx catalyzed the conversion of glucose into H₂O₂, resulting in the production of ˙OH by delivering H₂O₂in situ and lowering the pH level in the infection site. Thanks to the proximity of GOx and Pd–Ru NS, the substrate transfer could happen much faster, and the reaction efficiency was raised. Its self-activated cascade reaction mechanism thus showed exceptional antibacterial properties both in vitro and in vivo (Fig. 5d).

The CAT-like enzyme activity was also present in noble metal nanoparticles. Our team created a two-dimensional Pd@Au bimetallic core-shell nanostructure (TPAN) to encourage O₂ synthesis inside malignancies.¹⁰⁰ To combat tumor hypoxia over the long term, TPAN might consistently catalyze endogenous H₂O₂ and constantly generate O₂. Moreover, surface plasmon resonance was initiated by the NIR-II laser, which improved the catalytic activity of TPAN toward H₂O₂. TPAN was given X-ray, photothermal, and photoacoustic imaging (PAI) capabilities for precise imaging-guided cancer therapy and minimizing the side effects induced by X-ray irradiation by using high-Z elements, higher NIR-II absorption, and excellent photothermal efficiency.

4. Other transition metals-based nanomaterials

Besides Fe, Mn, and noble metals mentioned above, other transition metals-based nanomaterials were also chosen for tumor therapies via nanozymes.^101–103 In 2016, Wang and partners constructed a series of ultra-thin 2D-nanosheets through template synthesis by using Fe-TCPP (Fe^III tetra(4-carboxyphenyl)porphine chloride) as the precursor.¹⁰⁴ Three metals (Co, Cu, and Zn) were prepared for a series of 2D nanosheets (M-TCPP(Fe), M = Co, Cu, Zn). The obtained nanosheets showed comparable CAT-like activity against heme.

Li and coworkers developed an endogenous H₂S-activated Cu-MOF (HKUST-1) with HRP-like activity for colon cancer therapy.¹⁰⁵ In normal tissues, the catalysis activity of HKUST-1 was “OFF”, and no obvious NIR adsorption was observed. When nanoparticles reached colon tumor areas that expressed a high concentration of H₂S, HKUST-1 was activated to “ON” by the in situ production of photoactive CuS. The HRP mimicking activity endowed HKUST-1 with the ability to effectively convert H₂O₂ into ˙OH for CDT (Fig. 6a). Distinct from previous theranostics systems, this switch relies on the endogenous biomarker from the TME and has significant potential for accurate diagnosis, minimum invasion, and perhaps clinical translation.¹⁰⁵ Guo and colleagues developed a new paradigm of therapies for converting malignancies with overexpressed aberrant amounts of cholesterol into the highly cytotoxic ˙OH by using the cascade catalysis of the natural enzyme (cholesterol oxidase, COD) and nanozyme (Cu²⁺-modified Zr-MOFs) (Fig. 6b).¹⁰⁶

5. Other nanomaterials

Because of their biocompatibility and easy modification, carbon-based nanomaterials have become a promising tool for bioengineering applications. Various carbon-based nanomaterials have also been widely discovered for enzyme-like functions, including graphene oxide,¹⁰⁷ carbon nanodots,^108,109 carbon nanotubes,¹¹⁰etc. Among them, carbon nanodots were widely used as fluorescent probes with tunable optical properties. Dehvari and coworkers synthesized Mn-doped carbon nanodots for ROS scavenging via a CAT-like process to reduce oxidative stress damage along with fluorescence and MRI imaging¹¹¹ (Fig. 7a).

The ability to control enzyme activity in real-time has provided researchers with new insights into the physiological processes in which the enzymes are involved. The use of semiconducting polymer nanoparticles (SPN) in optical imaging and phototherapy has been made possible. For imaging-guided chemo-dynamic therapy, our team developed an integrated semiconducting polymer nanosystem (SPN) with ROS-correlated chemiluminescence.¹¹³ Hemin was added to our nanosystem, improving both chemiluminescence imaging (CL) and CDT, which produced highly toxic ROS under pathological situations because of the biological features of hemin. Significantly, our nanosystem was able to create an outstanding correlation between the CL imaging intensity and the inhibition rate of cancer cells following CDT in vitro and in vivo, as well as a relationship between CL intensity and the output of ROS in solution. Because of this association, our nano-platform might be able to assess the effectiveness of CDT at an early stage of therapy (Fig. 7b).

Besides singular nanomaterials, nanohybrid materials were also reported as nanozymes. Two or more kinds of nanomaterials or natural enzymes were combined to treat tumors with multiple enzyme-like activities. Inspired by the biomineralization process, our group developed a similar nanozyme@enzyme system for tumor therapy by self-cycling nutrition depletion and synergistic catalysis.¹¹²

The high catalytic ability of GOD complemented the high stability of nanozyme Cu_3+x(PO₄)₂. In this cascade catalysis system, GOD could catalyze glucose to produce H₂O₂, which further accelerated the following reaction of Cu_3+x(PO₄)₂ for ˙OH/¹O₂ generation and GSH consumption. This proposed nanozyme@enzyme symbiotic system may successfully inhibit the growth of tumors, offering a new research approach for tumor therapy (Fig. 6c and d). A four-in-one self-cascade nanohybrid with POD, CAT, and GPx-like enzyme-like activities was applied to boost ferroptosis stress for tumor therapy.¹¹⁴ This nanozyme was fabricated with MnO₂ nanodots, bovine serum albumin, and Au nanoclusters. Ferroptosis stress was enhanced due to the hypoxia relief (O₂ generation through a CAT-like process), oxidation of lipids (ROS generated from a POD-like process), and GSH depletion (GPx-like process).

The cytotoxicity of nanozymes in tumor therapy

Nanozymes are widely used as nanotheranostics in tumor therapy that utilizes the high catalytic activity.^115–117 The unique tumor microenvironment (high H₂O₂ and GSH concentration, low pH, etc.) endows the nanozymes with activatable catalytic ability.¹¹⁸

For POD-like and CAT-like nanozymes, the key to tumor therapy is the concentration of intratumoral H₂O₂.^119–123 Under the catalysis of nanomaterials, the excess H₂O₂ in tumor areas can be transformed into hydroxyl radicals (˙OH) or O₂ higher cytotoxicity to kill tumor cells directly or treat cancers along with other treatments (photodynamic, etc.) by supplementing local oxygen to boost the therapeutic effects. Thus, the catalytic activity of nanozymes is locked in normal tissues and activated in tumor areas. This kind of concentration-dependent catalysis could reduce the side effects on healthy tissues during the treatment. Glutathione peroxidase (GSH-Px) is a kind of enzyme that can convert GSH into GSSG.⁸⁶ Some of the nanomaterials can utilize GSH-Px-like activity to reduce the deoxidation ability to promote tumor therapy, especially ferroptosis. Without the high concentration of GSH, normal cells cannot activate the catalysis to prevent severe side effects.

Cao and coworkers developed a nanozyme system by using starvation, ferroptosis, and prodrug therapy.⁹⁵ A prodrug banoxantrone dihydrochloride (AQ4N) that can be converted into a cytotoxic DNA intercalator (AQ4) by the hypoxia condition in tumor tissues was conjugated with the Cu–Ag nanoparticles that endowed the nanozyme with high specificity towards tumor tissues, thus minimizing the off-target side-effects because of both hypoxia responsiveness and acidity favored prodrug release. Qin and colleagues used the Fe₃O₄@Cu_1.77Se nanozyme to treat tumors.¹²⁴ They chose gelatin, a polypeptide that can be degraded by matrix metalloproteinases (MMPs) to modify the nanozyme that can be degraded in tumor areas due to the overexpression of MMPs in tumors. Thus, the catalytic ability could be activated in tumor cells, while the catalysis in normal cells was suppressed.

In summary, in the development of nanozymes for biomedical applications, especially in tumor therapy, it is necessary to have high cytotoxicity in tumor cells and lower toxicity in normal tissues to avoid side effects. The current research is focused on strategies that can be utilized to regulate the catalytic activity of nanozymes in tumor microenvironments after reactions with tumor-specific pH value, GSH, H₂O₂, enzymes, etc.

Evaluating the catalytic activity of nanozymes in living systems

As we illustrated before, the therapeutic efficacy of nanozymes is related to their catalytic abilities, which are dependent on their catalytic activity. However, the complex tumor microenvironment affects the reactivity of nanozymes, causing huge differences in enzyme activities in vitro and in vivo. Moreover, the current methods for evaluating the enzyme activity cannot provide feedback for real-time information and are complicated to operate. This drawback of not being able to monitor the processes of nanozymes and evaluate the dosage may lead to severe side effects due to overdue usage and the imprecise targeting of nanozymes. Thus, the development of real-time imaging tools is quite necessary for monitoring the therapy processes.

In general, nanozymes play important roles that convert endogenous H₂O₂, O₂, and GSH into various ROS that influence the redox balance in tumor cells. Nanozymes have also been designed as O₂ generators to relieve tumor hypoxia via a CAT-like procedure. When the reaction occurs in vitro, only a few physical-chemical conditions affect the reaction efficiency, such as pH, temperature, and the concentration of catalytic substrates. However, the microenvironment in tumor cells is intricate. Various physiological conditions and chemical factors can create an interconnected and interactional balance in tumor cells. The influencing factors in vivo are complicated and complex. Thus, the catalyzing activity of nanozymes is quite different from that in vitro, and effective evaluation tools are needed in the biomedical applications of nanozymes for grasping the therapeutic information in real-time in vivo.

Imaging methods for evaluating the efficiency of nanozymes have been developed and reported, including fluorescent probes, chemiluminescent probes, magnetic resonance imaging (MRI) contrast agents, and photoacoustic imaging probes. Including our group, scientists all over the world have published over 4000 papers on this field in the past five years. We have reviewed the representative and constructive works from among those papers, and we hope that we can provide some novel viewpoints for other researchers regarding the latest advances in the imaging-monitoring of nanozymes (Fig. 8).

1. Fluorescent probes

Fluorescence imaging is used to precisely track the position of tumors or treatment processes, allowing for long-term biological distribution tracking and sensitive detections. Fluorescence is thus used in certain workable ways for the sensitive and real-time imaging of intratumoral ROS levels to track catalytic activities. Fluorescence imaging is also a practical and economical method for treatment monitoring because it is the most prevalent and significant phenomenon in luminescence imaging. The optical characteristics and biodistribution of fluorescence imaging reagents can be easily modified to meet a variety of needs, including monitoring ROS throughout treatments.¹²⁵

A unique Fenton-based nanotheranostics (NQ-Cy@Fe&GOD) method that creatively combined magnetic resonance imaging and dual-channel near-infrared fluorescence signals to reveal intratumoral ˙OH-mediated therapy was described by Ma and colleagues.⁴⁴ The fluorescence probe NQ-Cy (exhibiting dual fluorescence signals and two excitation channels) and iron oxide nanoparticles that contained glucose oxidase were co-loaded into the hydrophobic inner cavity of the amphiphilic DSPE-PEG to create a complete novel nanozyme (NQ-Cy@Fe&GOD). The MRI signal of IONPs was used for detailing the dose and biodistribution of the nanozyme. After the disassembly, a turn-on NIR fluorescence signal at 830 nm was observed when the nanozyme was internalized by the targeted cancer cells. Then, the GOD release started the oxidation processes, which produced a significant increase in H₂O₂in situ. Following the nanosystem breakdown and the release of INOPs, an amount of lethal ˙OH was produced through IONPs-mediated Fenton reactions. This cascade catalysis process led to the overexpression of NQO1, which was then detected at 650 nm by another fluorescent emission channel of NQ-Cy. It is of significance that this Fenton-based nanocomposite can intricately connect MRI and dual-channel NIR fluorescence imaging to achieve several advantages including mapping the biodistribution and dose by MRI, monitoring ˙OH generation and therapeutic effects via dual-channel fluorescence, obtaining real-time feedback of catalytic activities of the intratumoral Fenton reaction (Fig. 9).

A biocompatible polymer was used by Qi and coworkers to create an enzyme-catalyzed atomic-transfer radical polymerization technique (ATRPase).¹²⁶ Following the coordination of the amino/acid groups with the Fe^II ions, metal-coordinated polymeric nano-gels (MPGs) were created, and the resulting complex displayed effective multiple enzyme-like activities (SOD and POD). In the hydrophilic networks of MPGs, where Fe^II ions are mono-atomic and widely dispersed, they can act as a crosslinker for the gel network, and the active centre of enzyme mimics with excellent reactive efficacy. The catalysis of MPGs was successfully used in tumor-bearing mice to achieve effective ROS-responsive fluorescence imaging in response to the tumor overexpression of high-level superoxide free radicals (O₂˙⁻). MPGs can realize the optimized interactions between the active centres and substrates in the stable gel network by fully considering the structure–function relationships that resulted in excellent enzyme-mimic catalyzing effects for ROS-responsive fluorescence imaging for imaging-guided monitoring (Fig. 10).

2. Chemiluminescent probes

The interference of tissue autofluorescence can be eliminated by using optical imaging techniques without excitation light, e.g., chemiluminescence (CL) and bioluminescence. However, the expression of luciferase is the only factor that restricts the use of bioluminescent probes. CL has advantages such as no autofluorescence, greater signal-to-noise ratio, and higher sensitivity due to its requirement of specific chemical reactions to stimulate luminescent reporters. Thus, chemiluminescence can be used to develop imaging probes to detect biological and chemical biomarkers during the catalytic processes of nanozymes via the unique chemical reactions between CL substrates and the targets. Although there are no reports that chemiluminescent probes could directly detect the catalytic activity of nanozymes, chemiluminescence could be considered a potential method for monitoring the chemical and biological markers to provide feedback on the catalytic activity.

Our team created a series of CL probes with the ability to track the activity of nanozymes to detect ROS during tumor therapy. We created a therapeutic and monitoring device that is ROS-activated using extremely thin Mn-oxide (MnOx) nanosheets and semiconducting polymer nanoplatforms (SPN).¹²⁷ (Fig. 11). Importantly, the given ultrathin MnOx demonstrated the ability to produce ¹O₂ when acidity was activated, demonstrating a unique chemo-dynamic process. Moreover, the thiophene-based SPNs were stimulated to emit CL by the ultrathin MnOx nanosheets. In addition, the reaction between ¹O₂ and the thiophene units in the SPNs energized them to emit near-infrared (NIR) CL, which considerably increased the yield of ¹O₂. The MnOx-SPNs system produced good in vitro and in vivo chemodynamic therapeutic results under the activation of intrinsic acidity in tumors. We also achieved the chemiluminescence of O-pentacene, which was sensitive to ONOO⁻, based on embedding O-pentacene and a near-infrared SPN (PCPDTBT) into the nanoparticles using nanoprecipitation.¹²⁸ The obtained nanoprobe demonstrated very sensitive, targeted, and quick response detection by a particular reaction with ONOO⁻. This nanoprobe was able to emit near-infrared chemiluminescence signals for penetrating deep tissues because of the chemiluminescence resonance energy transfer (CRET) process. Also, it was successfully applied to visualize endogenous ONOO⁻via CL in vivo in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models.

As mentioned, we created an SPN, as shown in the previous chapter, which not only produced highly hazardous ROS but also emitted ROS-correlated chemiluminescence signals.¹¹³ Importantly, hemin doping can considerably increase the effectiveness of both chemiluminescence and CDT. The ROS-dependent CL of SPN enables the optical monitoring of ROS formation within a tumor during CDT. It is significant to note that SPN establishes a strong association between tumor inhibition rates both in vitro and in vivo, and chemiluminescence intensity levels. Because of the enabled CL-imaging-monitored CDT, our nanoplatform is the first intelligent method that can do this, and it could evaluate therapeutic responsiveness and forecast treatment outcomes at an early stage (Fig. 7b).

3. Photoacoustic probes

Photoacoustic imaging (PAI) is a noninvasive biomedical imaging technology for in vivo imaging.¹²⁹ Different from other imaging methods, due to the photoacoustic effect, PAI is viewed as a hybrid in terms of imaging technology that combines optical excitation and ultrasonic detection. Due to its superior spatial resolution and deeper tissue penetration as compared to fluorescence imaging, PAI provides volumetric information about the distribution of PA agents with ultrasonic resolution.^130,131

As mentioned in the previous sections, Sun developed a Pd/Pt-based CAT-like nanozyme.⁹⁸ The obtained nanozyme showed excellent PA signals under NIR laser irradiation, which provided PAI as a useful tool to monitor the distribution and tumor aggregation of nanozymes (Fig. 5c). Zhang and coworkers introduced Mn into the MoO_x semiconductor nanocrystal by thermal injection to create a composite nanozyme (MnMoO_X) for TME-activated cascade catalytic anticancer treatment that is directly monitored by tri-modal imaging.¹³² The introduction of Mn enhanced the NIR-II absorption of the nanozyme, bringing out a higher PTT conversion efficiency. Due to this characteristic, the MoO_X nanozyme also made an outstanding PTT agent, contrast agent, and photoacoustic signal. The use of Mn in magnetic resonance imaging was expanded by its inherent paramagnetic characteristics. As a result of the many flaws, MnMoO_X displays good catalytic reactions toward the TME, leading to effective POD and CAT-like activities. The cyclic response of MoOx is encouraged by the Fenton-like reaction between Mn²⁺ and H₂O₂ in the TME, which also improves enzymatic kinetics. To counteract the oxidative stress of cancer cells, high-valence manganese, and molybdenum also consume GSH in tumor cells. The proposed MnMoO_X system is easily excreted through the metabolic pathway, rapidly degrades, and loses its enzymatic activity in the physiological environment and is rather stable in acidic conditions. The MnMoO_X nanozyme has numerous flaws and can be used to remove tumors.

According to Wang, a polyoxometalate complex-based multifunctional nanozyme with GSH-Px and CAT activity (Ox-POM@Cu) was designed and synthesized.¹³³ Doping with Cu increased the concentration of carriers, sped up electron transfer, and improved conductivity, resulting in the development of oxygen vacancies in Ox-POM@Cu, which would further improve the redox activity of the nanozyme to perform as an effective catalyst. In the presence of endogenous GSH, Ox-POM@Cu demonstrated catalytic properties, quickly oxidizing it to glutathione disulfide and consuming the available GSH.

Some Mo⁶⁺ in Ox-POM@Cu is converted to Mo⁵⁺ by reduction, and the weakly acidic environment of the tumor is where the Mo⁶⁺/Mo⁵⁺ POM is created. POM displayed high NIR-II absorption, which endowed it with NIR-II PAI and activated PTT. The Cu⁺-enzyme then catalyzed H₂O₂ through a Fenton-like reaction in the TME to quickly produce ˙OH. At the same time, the GSH depletion can reduce the redox consumption of obtained ˙OH to maintain its concentration, which further strengthened the CDT impact against the tumor. Due to the low GSH and H₂O₂ contents in normal tissue, the described processes barely occurred. As a result, Ox-POM@Cu is inert to healthy tissues and the catalytic activity of the nanozyme only emerges in the TME (Fig. 12).

In a recent study, our team described an oxidase-like nanozyme (MSPN) by utilizing a semiconducting polymer combined with manganese that could realize efficient cancer therapy and self-reporting imaging of catalytic activity.⁸⁹ For the catalytic therapy of cancer, MnOx functioned as an acid-switchable oxidase-like nanozyme because of ROS production. Due to the efficient fluorescence resonance energy transfer (FRET) between PFODBT and ORM, MSPN displayed ratiometric NIR fluorescence and NIR PA dual-signal output when the bimodal probe ORM was destroyed by Mn^III generated from MnOx under acidic circumstances. Hence, MSPN generated oxidase-like activity and was further applied in cancer therapy in vivo and was able to achieve simultaneous ratiometric signals from dual-model NIRF-PA imaging to monitor the oxidase-catalysis in real-time (Fig. 13).

The major noninvasive imaging technique used in clinical diagnosis is magnetic resonance imaging (MRI). It can provide high spatial resolution for anatomical and physiological data. MRI, with good soft tissue contrast and deep tissue penetration, has allowed for the diagnosis and monitoring of a variety of disorders.^134,135

A core–shell nanozyme (Fe₃O₄@MnO₂) was created and put to use in combination therapy with GOX, according to Lyu.¹³⁶ The Fe₃O₄ core is a highly studied nanoparticle that can target the tumor and initiate the Fenton reaction in acidic conditions to generate enormous amounts of ROS. The intracellular excess H₂O₂ and the H₂O₂ created through the GOX in the presence of glucose were converted into a significant amount of oxygen, which relieved the hypoxia and repaired the DNA radicals. More importantly, the MnO₂ shell oxidized the GSH to GSSG, which inhibited the natural ability to repair itself after RT. During the above processes, high-valence manganese ions (Mn⁴⁺) were reduced to low-valence free Mn²⁺ ions, which served as a T₁-weighed MRI contrast agent while the Fe₃O₄ core was a superior T₂-MRI contrast agent. Thus, Fe₃O₄@MnO₂ can exhibit enhanced imaging signals in T₁ and T₂-weighed dual-modal MRI. Considering that Mn and Fe are daily required trace elements, Fe₃O₄@MnO₂ offers relatively high biosafety. As a result of the remarkable improvement in the hypoxia relief and repair inhibition, the combination of Fe₃O₄@MnO₂ and GOX was demonstrated as favorable for radiation therapy (RT), as well as reducing the dose for normal adjacent tissues.

Our team coated the PFOB nanodrops with a MnOx shell and then modified them with PEG to create a core-shell nanostructure (PFOB@MnOx, PM-CS NPs).⁸⁸ Mn ions were released in the presence of GSH and acidity to produce singlet oxygen (¹O₂) and hydroxyl radicals (˙OH). MnOx functioned as a GSH scavenger to consume antioxidants in tumor regions. PFOB was able to supplement a significant quantity of O₂ to increase lipid oxidate (LPO) levels by boosting ROS production and GSH depletion. It is interesting to note that the addition of oxygen directly reduced tumor hypoxia and suppressed the production of HIF-1α, which could reduce intracellular lipid droplet storage and increase the availability of free PUFAs for lipid peroxidation (Fig. 14a). Moreover, MnOx dampened the ¹⁹F MRI signal of the PFOB core due to paramagnetic relaxation enhancement (PRE) effects. Due to the decreased PRE effects, the ¹⁹F MRI signal could be activated by acidity or GSH. The release of Mn from PM-CS NPs was closely correlated with the activatable signal of T₁/T₂-MRI during the same period. For imaging-guided ferroptosis therapy, the activatable “hot-spot” ¹⁹F MRI with T₁/T₂-MRI contrast is a potential technique. The combination of chemical pathways (increasing ROS, depleting GSH, and boosting LPO) and biological pathways (inhibiting HIF-1α, destroying lipid droplets, supplementing more PUFAs, and upregulating ACSL4) allowed PM-CS NPs to overcome hypoxia-induced ferroptosis resistance, leading to effective and targeted ferroptosis-based anticancer outcomes in cancer cells and tumor-bearing mice (Fig. 14b).

Our team created another activatable nonferrous ferroptosis agent for MRI monitoring and ferroptosis-based tumor therapy.⁸⁷ Multivalent Mn was doped into a Pt skeleton to create a unique ternary nanoalloy (PtWMn) that served as an effective Mn reservoir. This allowed for the efficient storage, distribution, and release of Mn ions. The PtWMn nanoalloy was then coated with a pH-responsive polymer as a gatekeeper to create a pH-responsive nano-platform (R-PtWMn). When stimulated by acidity, Mn ions with multiple valences burst out from the PtWMn structure and performed a variety of enzyme-like functions (such as oxidase, horse radish peroxidase, glutathione peroxidase, and catalase). Due to the Mn ions’ accumulation, ROS creation, continuous O₂ production, GSH depletion, and increased lipid peroxidation, the catalytic activity of R-PtWMn might therefore be “switched on” for effective ferroptosis therapy. However, under physiological circumstances, R-PtWMn only released a small number of Mn ions, which silenced the catalytic activity and held tremendous potential for lowering the collateral cytotoxicity to healthy tissue. Moreover, Mn release-correlated T₁/T₂ high magnetic field MRI signals from antiferromagnetic R-PtWMn demonstrated the ability to monitor the beginning of nonferrous ferroptosis using both dual-mode and high-field MRI (Fig. 14c).

Summary and perspectives

Dozens of nanozymes have been created in the last decade by employing various nanomaterials. In addition to the theoretical mechanisms, fundamental ideas, and metrics for gauging their performance, extraordinary advancements have been made in the biomedical applications of nanozymes. The biomedical applications of nanozymes originated from the relationship between the specific physicochemical features of nanomaterials and enzyme-mimicking catalytic activities. It is vital to use imaging techniques to precisely assess the real-time catalysis levels in vivo to depict the real-time reaction processes. The creation and innovation of nanozymes have been outlined in this review, and the path of the nanozyme activity during the progress of cancer therapy was also discussed as a crucial aspect of nanozymes (Table 2). We have enlisted MR imaging, photoacoustic imaging, fluorescence imaging, and chemiluminescence imaging to illustrate the targets in monitoring nanozymes. These tumor imaging techniques could also be utilized to assess the therapeutic response following treatment and provide up-to-date information to direct accurate dose injection and avoid inaccurate dose injection to get the best possible therapeutic results.

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Although applications of various nanozymes have been booming over the past decade and some of them involved imaging methods, there is still a lack of appropriate real-time monitoring tools. There are also some drawbacks that impede the development of imaging methods. Firstly, the influencing factors of imaging probes or contrast agents are simple, singular, and independent in solution and in vitro. However, the microenvironment in vivo is complicated and involves multiple factors. These complex and numerous factors influence the imaging results and give false signals to disturb the monitoring of the nanozymes’ activities. Secondly, the higher activity of nanozymes is demanded to realize effective therapy effects, which brings about the new problem of imaging-guided therapy. The existing probes may be disrupted and destroyed due to the boosted ROS generation. On the contrary, the introduction of molecular probes or contrast agents may hamper the activity of nanozymes. However, the separation of theranostic agents and imaging probes could not precisely provide feedback for real-time signals in vivo. Therefore, more investigations and new insights to bring about powerful tools or new thoughts are needed to solve these problems. Although new tactics for monitoring nanozymes have made some headway, they are still in the early stages. To enhance the imaging probes, inorganic or organic elements might be mixed to complement each other. Multi-modality imaging may also be used for sensitive imaging to provide additional information about nanozymes and their specific features. Moreover, the non-specific nanozyme may have adverse effects on healthy tissues that are not reversible. Hence, there is an increasing need for the creation of stimuli-activated nanozymes with real-time imaging capabilities that are activated by specific elements during enzyme-mimicking methods.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by National Natural Science Foundation of China (grants 21974039, U21A20287, 22234003, 21977027), the Science and Technology Project of Hunan Province (2020RC3022; 2020SK2014), Shenzhen Science and Technology Program (JCYJ20210324140205013), China Postdoctoral Science Foundation (2022M711106, 2022TQ0101).

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