细胞内原位纳米催化在疾病诊断与治疗中的应用

张恩东，吕凤婷，黄一鸣，白昊天，王 树

（北京分子科学国家研究中心,有机固体院重点实验室,中国科学院化学研究所,北京 100190）

1 Introduction

In recent years，a variety of catalysts have been broadly utilized in industry，medicine，biology，and other fields for their superior catalytic activity and substrate specificity［1—4］.Compared to the harsh catalytic conditions for traditional chemical catalysts（high temperature and pressure，harmful solvents，and extreme pH），natural enzymes catalyze the transformation of biomolecules under relatively mild conditions，which are suitable for catalysis in the physiological environment［5］.Synthesis of enantioselective drugs in cascade reactions also needs catalytic enzymes with high specificity and efficiency to afford complex molecules in biomedicine.Development of sited-spot mutagenesis［6—8］and directed evolution［9］technologies have enabled the personalized design of enzymes utilized for the catalysis of specific and reactive substrates.Besides，high yield production of enzymes broadens their applications in the fields of synthetic biology in combination with fermentation technology.For the exploration of complex metabolic pathways in cells，gene editing technology is used to express exogenous proteases in cells［10—16］.

Although several natural biological enzymes are expected to become therapeutic proteins and provide a catalytic platform for the treatment of a variety of diseases［17］，many obstacles limit their further applications.High cost，poor stability，unstable catalytic activity，and difficult recycling constitute major issues that limit the application of natural enzymes in biosensors，environmental protection，biomedicine，and other fields［18］.In addition，the inefficient transduction of natural enzymes into cells hinders the regulation of intracellular metabolic activities and physiological processes［19］.With the increasing research of biomimetic materials inspired by nature，researchers are committed to designing and constructing catalytic materials with new functions and satisfactory cost，stability，and yield to break through the limitations of natural enzymes in various fields［20，21］.In recent years，advances in nanotechnology，biotechnology，and computer science have provided powerful tools for the development of high performance nanocatalysts that simulate intracellular metabolic activities due to their unique physical and chemical properties［22，23］.

Since the discovery of peroxidase（POD）-mimic Fe3O4NPs，nanocatalysts have been greatly developed，with adjustable catalytic activity and type，involving intracellular catalytic reactions for disease diagnosis and treatment［24］.These nanocatalysts showed high catalytic activity in a multienzyme mimetic manner and low cost with simple preparation processes.High stability allows nanocatalystsin vivostudy through systemic administration.Meanwhile，robust nanocatalysts have large surface areas to provide multiple modifiable sites to be engineered with functional groups.For example，the nanocatalyst is functionalized with targeting groups，which plays a guiding role in reducing unwanted toxicity and avoiding off-target effects with unnecessary accumulation［25］.Altogether，nanocatalysts are considered one of the most potent candidates to realizein situcatalytic reactions of regulating cellular metabolism for disease diagnosis and treatment.In this review，the developed nanocatalysts are briefly introduced first，particularly utilized for sensing and therapeutics of diseases（Fig.1）.Then，the examples of prospective nanocatalysts for intracellular catalytic reactions are demonstrated and their potential biomedical applications are outlooked.

Fig.1 Diagnostic imaging and therapeutic effects of nanocatalysts by intracellular in situ catalytic reactions

2 Sensing and Imaging for Diseases

Accurate diagnosis provides valuable information to guide the optimized strategy of prevention and treatment of diseases in their early stages［26—28］.Towards this end，nanocatalysts can be introduced to catalyze chemical reactions in response to the microenvironment of lesion sites.Products of the catalyzed reactions would allow accurate and selective imagingin situto discriminate lesion sites and healthy tissues.For example，amorphous calcium phosphate was utilized as a robust shield that encapsulated exogenous natural enzymes to form nanoparticles for detecting cancer cells by intracellular catalysis［29］.Vigorous metabolism of cancer cells showed abnormally higher level of glucose that propelled the encapsulated GOx for reactive oxygen species（ROS）generation.When ROS triggered hydrolysis of the ROS-responsive probe DCFH，fluorescence was observed inside cancer cells.

However，in the complex and variable cellular environment，the high catalytic activity of immobilized natural enzymes remained difficult in intracellular catalysis.Therefore，magnetoferritin nanoparticles were designed by encapsulation of iron oxide nanoparticles into recombinant human ferritin heavy chain（rhFTH）for targeting and imaging of tumoral tissues［30］.The rhFTH shell without any modified targeting ligands showed a distinguished homing ability to living tumor cellsin vitroandin vivo.In virtue of highly expressed H2O2in tumor cells，iron oxide nanoparticles were able to catalyze the oxidation of tetramethylbenzidine or diazoaminobenzene into blue-colored products.Magnetoferritin nanocatalyst with high sensitivity and specificity could serve as a diagnostic tool for guiding cancer diagnosis.

Wanget al.［31］designed an artificial enzyme PTT-SGH that responded to the high level of ROS and selfassembled into nanocatalysts，which catalyzed intracellular ester hydrolysis reactions.The multi-thiol molecular backbone was modified with imidazole groups and cross-linked into nanoparticles under high intracellular level of ROS in A549 cancer cells.In cross-linked PTT-SGH，imidazole groups in proximity formed a catalytic domain，in which proton transfer occurred for hydrolysis of the active ester to restore the fluorescence of nonfluorescent ester-caged fluorescein precursor.The whole process was illustrated in Fig.2.It was verified that nonfluorescent ester-caged precursor would not be influenced by other biomolecules in the intracellular environment.In co-culture of cancer cells and normal cells，the artificial enzyme was internalized by both cells，but only ROS-overexpressed cancer cells could catalyze the ester hydrolysis and turn on fluorescent probes.This artificial intracellular nanocatalyst provided a new view for visualizing tumor sitesin vivo.

Fig.2 Illustration of ROS-induced cross-linking of PTT-SGH and catalytic hydrolysis of ester-caged fluorophore FN and activate the turn-on fluorescence inside cancer cells for selective diagnosis(A),and confocal laser scanning microscopy(CLSM)images of distinguishing A549 cells in a mixed culture incubated with FN and PTT-GSH(B)(scale bars:30μm)[31]

In addition to fluorescence imaging，photoacoustic imaging is a powerful bioimaging modality，which captures photo-induced ultrasonic waves that can penetrate deep tissues.POD-like nanocatalyst graphene quantum dots（GQDzyme）can catalyze the production of contrast agent with near-infrared（NIR）absorption，which is highly effective in the diagnosis of nasopharyngeal carcinoma［32］.Both GQDzyme and 2，2′-azinobis-（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS）were loaded into biomimetic folic acid functionalized exosomes，which efficiently accumulated in nasopharyngeal carcinoma and selectively triggered catalytic photoacoustic imaging in the presence of intracellular H2O2.Compared with normal tissues，GQDzyme could sensitively detect the relatively increased hydrogen peroxide in nasopharyngeal carcinoma，which was due to the rapid metabolism of tumors by producing oxidized ABTS as a contrast agent.It is worth mentioning that the exosome as a carrier platform stabilized the nanocatalyst，prolonged the circulation time，and improved the accumulation in tumor tissue after intravenous injection.

3 Anti-cancer Treatment

Although commonly practiced in clinical treatment of cancer，tumor removal by surgery is still limited by the slow postoperative wound healing and incomplete clearance of cancer cells［33，34］.Emerging strategies such as photodynamic therapy and radiotherapy provide new perspectives for cancer treatment［35］，but the therapeutic efficiency depends on sufficient oxygen supply in tumor microenvironment（TME）.The unique hypoxia condition in TME of solid tumors presents a limitation to the efficiency of cancer treatments.In contrast，nanocatalysts used as enzyme mimics can catalyze intracellularin situcascade reactions to produce reactive oxygen species for chemodynamic therapy.The nanocatalyst exhibits significant catalytic and therapeutic effects on cancer cells and is not affected by TME with low side effects.

Liuet al.［36］designed and constructed a PtFe@Fe3O4nanocatalyst that overcame the disadvantage of hypoxic condition in the treatment of deep pancreatic tumors through the autocatalytic reaction of oxygen and ROS.The PtFe@Fe3O4nanocatalyst was prepared by the assembly of Fe3O4on the surface of PtFe nanorods，which showed the same catalytic activities as both POD and catalase（CAT）.Due to the overexpression of hydrogen peroxide（H2O2），it catalytically generated hydroxyl radical（·OH）from H2O2in situ，which was cytotoxic to cancer cells accompanied by regeneration of O2.In PtFe@Fe3O4mediated nanocatalyst catalytic reaction，electrons flowed between Fe3O4|PtFe@Fe3O4and PtFe|PtFe@Fe3O4that made sustaining production of O2and·OH.The PtFe@Fe3O4nanocatalyst was effectively internalized by pancreatic cancer cells and displayed efficient accumulation in solid tumors followed by anticancer effect.It was worth to mention that synergy photothermal therapy with a photo-enhanced effect occurred under NIR illumination based on surface plasmon resonance effect of Pt.Because the active center of nanocatalyst was partially shielded，the catalytic activity of nanocatalyst mainly depended on the surface layer of atoms.To overcome this disadvantage，Zhaoet al.［37］developed a metal-organic framework（MOF）-based copper hexacyanoferrate（Cu-HCF）singlesite nanocatalyst as glutathione oxidase（GSHOx）and POD to deplete the overexpressed GSH in cancer cells，resulting in cytotoxic·OH and increasing acidity.MOF provided a porous platform for active Cu，which was separated from other atoms，thus improving the catalytic efficiency.

Nanocatalysts can catalyze tumor-specific reactions to inhibit tumor activity，but selective catalytic therapy in cancer cells with low intracellular H2O2level is ineffective.Hence，glucose oxidase（GOx）was loaded with MOF-based Co-ferrocene（Co-Fc）nanocatalyst to form a cascade of catalytic system（Co-Fc@GOx）for effective tumor therapy［38］.With uptake of Co-Fc@GOx by cancer cells，the therapeutic cascade occurred when excess glucose in cells was catalyzed by GOx to produce H2O2.The upregulated H2O2provided enough substrate for Co-Fc nanocatalyst to generate cytotoxic·OH by Fenton effect.In addition，the multi-enzyme mimicking nanocatalyst was developed to incorporate catalytic activities of POD，CAT，superoxide dismutase（SOD）and GSHOx，thereby combined the synergistic chemodynamic，photodynamic，photothermal therapies［39］.The“all-in-one”nanocatalyst was doped by Fe3O4and Ag into the Bi2MoO6substrate，which simultaneously and continuously produced singlet oxygen（1O2）and·OH accompanied by reduced pH（Fig.3）.This self-replenishing nanocatalyst formed a catalytic cycle to continuously maintain sufficient intracellular oxygen and H2O2.H2O2was catalytically converted into cytotoxic·OH by nanocatalyst in a POD activity and cytotoxic1O2was generated from O2viachemodynamic transformation to kill cancer cells.

Fig.3 Schematic illustration of self-replenishing nanocatalyst Fe3O4/Ag/Bi2MoO6 in the catalytic cycle to continuously maintain sufficient intracellular oxygen for catalytic cancer therapy[39]

Guet al.［40］reported layered MoO3nanobelts as efficient nanozyme for tumor-specific photo-enhanced catalytic therapy.They constructed a glucose-responsive cascaded nanocatalytic reactor by loading GOx and chemotherapeutic drug tirapazamine on the surface of the MoS2nanozyme，which achieved self-enhanced chemo-catalytic therapy［41］.Furthermore，they constructed molybdenum disulfide/hafnium dioxide（MoS2/HfO2）dextran nanoradiosensitizer to suppress tumor by improving the irradiation effectiveness［42］.

For the aforementioned nanocatalysts，metals were used as active centers to catalyze the reaction in cancer cells.Metal-free nitrogen-doped carbon nanomaterials（N-CNMs）were also developed with the activities of oxidase（OXD）/POD or CAT/SOD depending on pH level［43］.After uptake by cancer cells，N-CNMs were located in lysosomes，which provided a low-pH microenvironment to promote the radical generation by N-CNMs in OXD/POD manner.At a neutral pH，N-CNMs were able to utilize O-·2and H2O2to restore the level of O2.The N-CNMs showed high-efficiency anticancer activities bothin vitroandin vivothrough intracellular catalysis.

Wanget al.［44］proposed nature-inspired nanothylakoids as a multimodal theranostic agent for cancer therapy bothin vitroandin vivo.The peroxidase-like catalytic activity of nanothylakoids was verified，which facilitated the oxidation of H2O2to the cytotoxic hydroxyl radical and thus caused efficient cell apoptosis.Combined with the photothermal/photodynamic effects，nanothylakoids extracted from spinach leaves holds great promise in multimodal cancer therapeutics.

In addition to chemodynamic therapy，nanoparticles were also used as boosters for enhanced photodynamic therapy or radiotherapy.For radiotherapy，Yanget al.［45］reported core-shell Au@MnO2nanoparticles that could catalytically supply complemental O2in hypoxia conditions.MnO2served as the catalyst of Fenton reaction to catalyze intracellular H2O2into O2for the improvement of intracellular mircroenvironment.Increased intracellular O2provided condition for Au which served as a radiotherapy sensitizer to absorb ionizing radiation for the catalysis of DNA into DNA radicals to kill cancer cells.Catalytic production of O2by MnO2could also promote the efficiency of photodynamic therapy.The nanocatalyst that combined chlorine e6 and PEG-modified MnO2improved the efficacy of tumor-specific photodynamic therapy［46］.Based on the prominent catalytic decomposition ability of noble metals on H2O2，monatomic ruthenium self-assembled into Mn3［Co（CN）6］2MOF through combined driving forces of electrostatic，coordination andπ-πstacking interactions，which showed a strong therapeutic effect on hypoxic tumor microenvironment［47］.Here，active monatomic ruthenium acted as a catalytic photosensitizer to generate ROS to kill cancer cells.

4 Anti-oxidative Stress Treatment

The expression of oxidase and antioxidant enzymes is to maintain balance in the catalytic redox cycle［48］.Once the antioxidant enzyme fails or its activity decreases，the redox cycle will suffocate，resulting in the accumulation of internal ROS and dysfunction of biomolecules，including the carbonylation of denatured proteins，the oxidation of lipids，and the degradation of nucleic acids.In fact，oxidative stress caused by the imbalance of intracellular oxidation and conduction conditions can lead to many diseases，such as inflammation，diabetes，cardiovascular disease，and cancer［49—51］.Although some antioxidant enzymes may restore functions，excessive cytotoxic ROS still exist，resulting in secondary damage to antioxidant enzymes.Therefore，stable nanocatalysts are induced to scavenge cytotoxic reactive oxygen species in cells to protect cells from oxidative stress.

V2O5nanowires were self-assembled with MnO2nanoparticles via the linkage of dopamine，which acted as glutathione POD，SOD，and CAT to prevent oxidative stress in cells［52］.It catalyzes the conversion of O-·2into H2O2in cells and finally stabilizes to water，thus reducing the level of ROS.Electron transfer involving H2O2and O2depleted ROS and protected cytochrome c from oxidative damage in mitochondria［53］.Multi-antioxidant enzyme nanocatalysts established cascade catalytic systems to promote the progress of reactions，which have distinct stability and catalytic activity［54］.Essential regulation and response made the output of intracellular catalytic reaction controllable in a mild state.

Gold nanoparticles have been shown to drive CAT-like reactions at low pH or antioxidase-like reactions at relatively high pH［55］.Quet al.［56］designed reversible“on-off”nanocatalysts to regulate intracellular ROS levels.Gold nanoparticles（AuNP）were used as the active nanocatalyst and loaded into the azobenzene-modified mesoporous silica，but the catalytic activity was switched off because the pores were blocked by cyclodextrin（CD）through host-guest interaction［57，58］.Under UV irradiation，azobenzene was transformed into its trans-isomer，resulting in dissociation from CD.It unblocked pores of silica and released AuNP to remove ROS in cells.Under different light conditions，AuNP can be re-encapsulated by the supramolecular assembly of CD and cis-azobenzene to achieve reversible regulation（Fig.4）.

Fig.4 Schematic illustration of light controlled intracellular gold nanocatalyst to regulate ROS scavenging behavior by supramolecular host-guest interaction(A)and the changes of the ROS level in MCF-7 cells with the different irradiation time under the condition of exogenous,and alternately irradiated with UV and visible light(B)[56]

The ratio of nicotinamide adenine dinucleotide（NAD+）/NADH is a symbol of the redox state of mammalian cells［59］.Wanget al.［60］reported a multifunctional nanocatalyst，ruthenium-coordinated oligo（p-phenylenevinylene）（OPV-Ru），which catalyzed the intracellular hydrogenation transfer from nicotinamide adenine dinucleotide（NAD+）to its reduced format NADH.OPV-Ru can form nanoparticles in water through hydrophobic interaction andπ-πstacking，which create a local microenvironment for enhancing the catalysis efficiency with a 15-fold enhancement of turnover frequency（TOF）in comparison to the dispersed catalytic center molecule.Moreover，the fluorescence of OPV moieties facilitates the visualization of the dynamic distribution and uptake behavior of these nanocatalysts.

In addition to metal-based nanocatalysts，fullerenes have also been identified as inorganic nanocatalysts with the ability to scavenge free radicals［61］.With the development of 2D carbon materials，graphdiyne has been reported as a peroxidase simulated nanocatalyst，which can catalyze tetramethylbenzidine into the oxidation state in the presence of H2O2［62］.Subsequently，bovine serum albumin-modified graphdiyne（BSAGDY）nanoparticles were introduced into normal cells for radiation protection by eliminating ROS produced during radiotherapy［63］.BSA-GDY effectively protected DNA from radiation damage and restored SOD and malondialdehyde in normal human umbilical vein endothelial cells（HUVEC）.BSA-GDY provided a promising method to reduce side effects and expands the application of radiotherapy in the treatment of a variety of diseases.

Meanwhile，nanocatalysts can remove the excess ROS to alleviate inflammatory symptoms.Weiet al.［64］reported a one-pot synthesized Pt@PCN222-Mn nanocatalyst for treating inflammatory bowel disease.In cascade reactions，Pt@PCN222-Mn first catalyzed the conversion of ROS to H2O2by SOD activity，then converted H2O2to water and oxygen by the CAT activity.Inflammatory cytokines decreased significantly due to the increase of ROS level in colon tissue.Cascaded nanocatalysts with multifunctional active sites have shown superior anti-inflammatory therapeutic effects on ulcerative colitis and Crohn′s disease.

5 New Development of Nanocatalysts as Potential Therapeutic Tools

Nanocatalysts can efficiently and selectively catalyze the reactions using endogenous molecules in cells for disease treatment［24］.Bioorthogonal reactions can be initiated by nanocatalysts to generate active moleculesin situ，reduce off-target cytotoxicity and functionalize catalysis in living cells for diagnostic imaging and treatment［65］.For example，POD-mimic nanocatalyst catalyzed H2O2-independent oxidation for imaging of living cells［66］.NiO exhibits excellent oxidase like activity，which is not limited to H2O2.Different from the acidic pH required by other artificial nanocatalysts similar to oxidase，NiO converted amplex red to oxidized fluorescent resorufin and emit fluorescence at physiological pH.Zimmermanet al.［67］reported that the“click”reaction of alkyne azide was catalyzed by a self-assembled nanocatalyst based on a single chain linear polymer，which coordinated with the copper catalytic centers.The Cu-containing nanocatalyst showed a high affinity for hydrophobic substrates at ppm level，and high catalytic efficiency to turn on fluorescence in living cells.In addition，the active catalytic center was replaced by tris（bipyridine）-ruthenium［Ru（bpy）3］complex and crosslinked with water-soluble polymer to form a new nanocatalyst（Fig.5）［68］.The Ru-complex catalyzed the conversion of azide to amino groups.In order to internalize the nanocatalyst，β-galactosidase was used as a shuttle to transport nanocatalysts into the endosomes.After cell uptake，the nanocatalysts andβ-galactosidase maintained the catalytic ability to restore the fluorescence of inactive fluorescent probes.Based on the work reported above，exogenous molecules were catalyzed in cells by artificial nanocatalysts in various types of reactions，providing access to the biochemical factory in cells，where small precursor substrates diffuse into cells and complex synthetic products can be manufactured as needed.

Fig.5 Preparation of dual nanocatalytic system with tris(bipyridine)-ruthenium[Ru(bpy)3]complex and β-galactosidase(A),schematic illustration of intracellular dual catalysis for fluorogenic reactions to restore the fluorescence of fluorescein and coumarin(B),and confocal of inactive fluorescent probes Hela cells which were treated with dual nanocatalysts(C)[68]

6 Summary and Outlook

In recent years，remarkable achievements have been made in the application of intracellular nanocatalysts.Robust nanocatalyst becomes a promising tool that can selectively catalyze endogenous molecules in living cells for a variety of potential applications，such as tumor imaging，cancer treatment，and antioxidant stress treatment.The introduced exogenous substrate molecules carry out different types of reactions under artificial nanocatalysts，including click reaction，oxidation reaction，and reduction reaction.Thein situcatalytic reactions by nanocatalysts not only generate functional customized molecules，but also reduce the off-target cytotoxicity caused by unwanted reactions in normal cells.However，nanocatalysts still face many challenges.Firstly，there is an urgent need to establish standards for various nanocatalysts for systematic research due to difficulties in the evaluation and quantification of different nanocatalysts.Secondly，nanocatalysts need to have high efficiency and versatility for broader applications includingin vitroimaging，in vivodiagnosis and treatments，which are limited by the incompatibility of multiple nanozymes and lack of stimuliresponsive nanocatalysts.Finally，biodistribution，pharmacokinetics，and metabolism of biocompatible functional nanocatalysts in organisms should be studied in depth，which is conducive to subsequent clinical translation.In conclusion，we believe that nanocatalysts will have great therapeutic potential in the future.