Pathogenesis-guided nanozymes: from design to therapy for gastrointestinal inflammation

3.1.1 SOD-like nanozyme

SOD is the primary cellular defence against superoxide radicals (${\mathrm{O}}_2^{-}$) and protects organisms from oxidative stress^[20]. ${\mathrm{O}}_2^{-}$ is the key element in the production of ROS that are produced as a byproduct of metabolic processes in living systems. In the presence of SOD, ${\mathrm{O}}_2^{-}$ generated during metabolism disproportionately are converted to hydrogen peroxide and oxygen (Equation 1)^[21].

Most SOD nanozymes consist of transition metals (Cu, Fe, Ce, etc) and elements such as N, O, C, and S. The mechanism of action of SOD relies on the cycling of the reduced and oxidized states of redox-active transition metals (eg, Cu and Mn) at their active sites (Equations 2 and 3)^[22,23]. In general, ${\mathrm{O}}_2^{-}$ consists of Brønsted bases with pK_b = 9.12^[24], and thus ${\mathrm{O}}_2^{-}$ can trap protons from H₂O to form HO₂⁻ and HO⁻. HO₂⁻ adsorption on the surfaces of gold, silver, palladium, and platinum triggers the conversion of HO₂⁻ to O₂ and H₂O₂^[20].

For example, CeO₂ nanozymes are well known SOD mimics^[25]. Changes in the oxidation state of CeO₂ produce oxygen vacancies in the lattice structure through the release of oxygen and electrons. Oxygen vacancies play a key role in displaying SOD-like activity, enabling CeO₂ nanozymes to take up or release oxygen. The presence of Ce³⁺ is a consequence of oxygen vacancies; thus, a high Ce³⁺/Ce⁴⁺ ratio provides more oxygen vacancies, which enhances SOD-like activity. The decomposition of ${\mathrm{O}}_2^{-}$ by CeO₂ nanozymes is shown in Figure 1 (SOD-part CeO₂).

C-dot nanozymes are also typical enzymes with SOD-like activities. Gao et al.^[26] revealed the surface state-dependent catalytic activity of C-dot SOD nanozymes by surface structural tuning and theoretical calculations. The hydroxyl and carboxyl groups of the C-dots bind superoxide anions, and the carbonyl groups oxidize superoxide anions, producing oxygen and reduced-state C-dots. The reduced C-dots are oxidized back into the initial state by another superoxide anion and produce H₂O₂. The carbonyl group of C-dot nanozymes is the catalytic site for SOD-like activity, and its proposed reaction pathway for the SOD-like catalytic cycle with and without hydroxyl group is shown in Figure 2A. The reasonable mechanism of SOD-like activity with C-dots as catalytic sites is shown in Figure 1 (SOD-part C-dots).

3.1.2 CAT-like nanozyme

CAT is an antioxidant enzyme containing iron porphyrin in its active site and is present in almost all organisms that come into contact with oxygen^[27]. CAT catalyzes the degradation of 2 hydrogen peroxide molecules to produce 2 water molecules and 1 oxygen molecule (Equation 4), thereby protecting tissues from potential oxidative damage. First, the iron porphyrin group of CAT (Fe³⁺) reacts with 1 hydrogen peroxide to form an iron porphyrin cation radical (Fe⁴⁺). The radical then degrades the hydrogen peroxide to produce water and oxygen molecules, while the Fe⁴⁺ is reduced to the Fe³⁺ state^[28]. When iron chelates are in action, H₂O₂ reacts with O₂ to form the harmful -OH, which occurs only when 2 H₂O₂ molecules encounter the CAT successively and hit its active site^[2].

To date, a series of metal-related nanozymes, such as platinum (Pt), gold (Au), CeO₂, and Mn₃O₄, have been shown to possess CAT-like activities^[29]. Although the full mechanism of CAT-like nanozymes has not been fully elucidated, 2 possible catalytic reaction pathways have been summarized based on the pattern of chemical bond breaking during H₂O₂ decomposition^[30].

The first catalytic reaction pathway is the preferential breaking of the H-O bond of H₂O₂. Pirmohamed et al.^[31] found that CeO₂ nanozyme exhibits CAT-like activity through redox reactions, and higher levels of Ce⁴⁺ would contribute to the catalytic activity. Subsequently, Celardo et al.^[32] further elucidated the antioxidant mechanism of CeO₂ nanozymes (Figure 1 CAT-part CeO₂). First, H₂O₂ was adsorbed on the CeO₂ surface, followed by the release of 2 protons and the production of O₂, while Ce⁴⁺ was reduced to Ce³⁺. Then, another H₂O₂ molecule binds to Ce³⁺ and produces H₂O, at which time Ce³⁺ is oxidized to Ce⁴⁺. In addition, Li et al.^[33] investigated the enzyme-like activities on the surfaces of Au (211), Au (110), and Au (111) under acidic and alkaline conditions by analyzing experimental data and density functional theory (DFT). Comparison of reaction energy barriers and adsorption energies showed that preadsorbed OH groups have a strong influence on H decomposition and catalyze the preferential breaking of H-O bonds by H₂O₂ (Figure 2B). A similar situation is observed for other metals (eg, Ag, Pt, and Pd).

The second catalytic reaction pathway is the preferential breaking of the O-O bond of H₂O₂. The CAT-like activity of metal oxide nanoparticles (NPs) is usually derived from ionic pairing, such as Fe²⁺/Fe³⁺, Co²⁺/Co³⁺, Mn³⁺/Mn⁴⁺, and Ni²⁺/Ni^3+[2]. The discovery of ferromagnetic nanoparticles with peroxidase-like activity was reported in 2007, and these are believed to be the first inorganic nanoparticles to be used as enzyme mimics for biomedical applications^[1]. Subsequently, in 2011, Nie’s group observed that ferritin-platinum nanoparticles (Pt-Ft) have CAT-like activity under alkaline and neutral pH conditions, and the decomposition process can be summarized in Figure 1 (CAT-part Pt-Ft)^[34]. In addition, Wang et al.^[35] prepared Pd@TiO₂ with lattice defects and cavities, and similarly demonstrated that H₂O₂ adsorbed on the surface of Pd@TiO₂ is more prone to break the O-O bond, and thus cleaved into 2 -OHs, which undergo a transition state to ultimately produce.

3.1.3 Glutathione peroxidase-like nanozyme

The peroxidase (POD) family is very large and most of the peroxidases are heme enzymes with iron protoporphyrin IX (protoheme) as a repair moiety, such as horseradish peroxidase, lignin peroxidase, and myeloperoxidase^[29]. Currently, much attention is being paid to peroxidases with selenium as the active center (glutathione peroxidase, GPx), which are involved in the termination of the ROS pathway, thereby reducing oxidative stress^[36]. The active site of GPx contains selenocysteine, where selenohydrin (ESeH) degrades H₂O₂ to H₂O by redox and is oxidized to form selenoic acid (ESeOH). ESeOH then undergoes a redox reaction with 2 reduced glutathione (GSH) and reverts to ESeH. In this redox cycle, the 2 GSH are oxidized to form 2 glutathione disulfides (GSSG)^[37]. GPx has been found to catalyze multisubstrate reactions through an ordered mechanism^[38] and a ping-pong mechanism^[39].

The ordered mechanism suggests that GPx binds to multiple substrates in a specific order and then releases the product when all substrates bind to GPx. Zhang et al.^[40] prepared selenium-containing pentapeptide-modified Au NPs (Au@SeH), and kinetic analyses showed that Au@SeH adsorbed an H₂O₂ molecule first, and then bound to 2 GSH molecules sequentially (Figure 1 GPx-part Au@SeH). In this process, Au@SeH catalyzed the reduction of H₂O₂ to H₂O by GSH (Figure 2C).

The ping-pong mechanism suggests that GPx binds or releases alternately with substrates and products. Huang et al.^[41] synthesized GO-Se NPs with more efficient GPx-like activity. Se reacts with H₂O₂ to form Se₂O₃ intermediates. The resulting intermediate catalyzes the conversion of GSH to GSSG, while the Se intermediate returns to its original state. The GSH reductase can then catalyze the conversion of GSSG to GSH with the aid of nicotinamide adenine dinucleotide phosphate. Then, Se will react with another molecule of H₂O₂ (Figure 1, GPx-part GO-Se NPs). Thus, the catalytic mechanism of GO-Se NPs follows the ping-pong mechanism of catalytic degradation of H₂O₂.

3.1.4 Oxidase-like nanozyme

Oxidase (OXD) is a class of enzymes that directly utilize O₂ as an electron acceptor to catalyze the oxidation of substrates^[42]. Certain nanomaterials (such as MnO₂, CeO₂, and CuO) can mimic the activity of natural oxidases, oxidizing specific substrates (such as TMB and DOPA) without the presence of H₂O₂, accompanied by colorimetric reactions or the generation of ROS. Their catalytic mechanism primarily involves 3 key steps. First, the surface of the nanomaterials adsorbs and activates O₂ through changes in the valence state of metal ions (e.g., Mn⁴⁺/Mn³⁺ or Cu²⁺/Cu⁺), generating ROS. Subsequently, these ROS attack substrate molecules (e.g., oxidizing colorless TMB to blue oxTMB), completing the colorimetric or fluorescent reaction. Finally, metal ions on the surface of the nanomaterials (e.g., Cu²⁺ and Mn⁴⁺) are continuously reduced and oxidized during the reaction, maintaining the catalytic cycle.

For example, Cheng et al.^[43] investigated the O₂-dependent catalytic behavior of CeO₂ and confirmed its OXD-type activity under the study conditions. In the reaction mechanism, O₂ molecules were adsorbed onto defect sites of nanoceria and converted to ${\mathrm{O}}_2^{-}$ under acidic conditions. As surface Ce⁴⁺ is reduced to Ce³⁺, TMB is oxidized to TMBX. As the primary intermediate, the in situ-generated ${\mathrm{O}}_2^{-}$ ultimately regenerates Ce⁴⁺ by oxidizing Ce³⁺ while producing water. Additionally, the oxidation of TMB can also be directly initiated by ${\mathrm{O}}_2^{-}$ (Figure 1 OXD-part CeO₂). The possible reaction mechanism of Mn₃O₄ nanoparticles proposed by Zhang and Huang^[44]. The transfer of manganese to O₂ electrons leads to the formation of ${\mathrm{O}}_2^{-}$, part of which undergoes a nonenzymatic or SOD-catalyzed disproportionation reaction to produce H₂O₂ and O₂. Subsequently, part of the generated H₂O₂ reacts with dissolved Mn²⁺ and decomposes into ·OH. Subsequently, the intermediate ·OH/${\mathrm{O}}_2^{-}$ and Mn³⁺ oxidize TMB, thereby forming the TMB–Mn₃O₄ nanoparticle system.

Other nanozymes similar to oxidases catalyze specific substrates. For example, Au nanozymes catalyze glucose similarly to glucose oxidase^[45], CuAg alloys^[46], and Cu₂O^[47] catalyze similarly to cytochrome c oxidase. Pt NPs catalyze similarly to catechol oxidase^[48]. Au nanorods/Pt nanodots and PtCu NPs catalyze similarly to iron oxidase^[49].

3.1.5 Other-like nanozyme

In addition to the oxidoreductase family, other biologically active nanocatalysts have been increasingly discovered, including hydrolase-like, isomerase-like, and lyase-like enzymes. For example, peptide-functionalized monolayer-protected gold clusters (Au MPCs) have been shown to exhibit simulated activity of nucleases, esterases, and silicases^[50]. Cerium nanoparticles (CeNPs) have also been found to exhibit phosphatase-like properties, capable of hydrolyzing the phosphate bonds of ATP, p-nitrophenyl phosphate, and o-phosphotyrosine^[51]. Magnetic CuFe₂O₄ exhibits intrinsic protease-like activity, capable of hydrolyzing bovine serum albumin and casein under physiological conditions^[52]. CdTe quantum dots (4.5 nm) can recognize GAT^ATC DNA sequences and induce light-triggered T^A phosphodiester bond cleavage^[53]. Cysteine-derived chiral carbon dots exhibit activity similar to topoisomerase I, mediating enantioselective topological rearrangement of supercoiled DNA^[54]. Additionally, some nanomaterials can catalyze biochemical reactions that natural enzymes cannot, such as MOF-Cu-catalyzed azide-alkyne cycloaddition^[55] and Pd nanoparticles-catalyzed hydrogenation of ·OH^[56].

3.1.6 Catalytic mechanisms and their function in gastrointestinal inflammation therapy

Nanozymes mimic the catalytic activities of multiple natural enzymes to establish a multitarget synergistic therapeutic system for GI. In ROS scavenging, different enzymatic activities coordinate to form cascading reactions. SOD-CAT cascade system achieves complete detoxification from superoxide anion to water, effectively breaking the vicious cycle of oxidative stress-inflammation. These antioxidant mechanisms suppress proinflammatory factor release by eliminating ROS signals required for NLRP3 inflammasome activation. They also promote macrophage polarization from M1 to M2 by regulating intracellular H₂O₂ levels, thereby restoring immune homeostasis. For mucosal repair, besides protecting TJ proteins through antioxidant effects, the OXD mimic enzyme actively promotes epithelial regeneration by modulating H₂O₂-mediated Nrf2 signaling pathways. GPx preserves cell membrane integrity by reducing lipid peroxides^[36]. For gut microbiota regulation, nanozymes restore microbial balance through multiple pathways: enhancing beneficial bacterial proliferation by improving the redox microenvironment, while achieving selective antibacterial effects via •OH radicals generated by POD activity or lysozyme-like activity. This synergistic mechanism-function-therapy framework positions nanozymes as an ideal therapeutic strategy for GI.

3.2.1 Size control

Nanoscale nanozymes can easily penetrate the TJs of epithelial cells to reach the inflamed area, while also increasing permeability at the site of inflammation and prolonging drug retention time. Smaller sized nanozymes tend to exhibit higher enzyme-like activity with higher surface area and volume ratios, which facilitates interaction with substrates^[57,58]. Baldim et al.^[59] reported that CeO₂ nanozymes with a size of ≈5 nm had the highest SOD-like activity, followed by ≈8 nm, ≈23 nm, and ≈28 nm, respectively. Suggesting that the size of the CeO₂ nanozymes is almost inversely proportional to the SOD-like activity, due to the fact that a decrease in the size of the particles leads to an increase in the Ce³⁺/Ce⁴⁺ ratio on the surface of the particles, which in turn increases the number of defects due to oxygen vacancy quantity. More oxygen vacancies provide more active sites for reaction with substrates^[60]. Shlapa et al.^[61] also showed that cerium oxide nanozymes with size of 4.2 nm also exhibited higher antioxidant activity as compared with size 14 nm (Figure 3A). Similarly, C-dot nanozymes with a size of 2 nm and a large specific surface area also provided abundant binding and active sites for the catalytic reaction, which led to the enhancement of SOD-like activity^[26].

Xia et al.^[62] explored the surface states and antioxidant activity of Ru NPs of different sizes and found that the proportion of the surface oxidized states increased significantly as the size of Ru NPs decreased. When the size was reduced to 2.0 nm, the surface oxidized Ru atoms of Ru NPs dominated, thus possessing an unprecedentedly boosted antioxidant activity as compared with medium-sized (≈3.9 nm) or large-sized counterparts (≈5.9 nm) that are mainly composed of surface metallic Ru atoms. The results suggest that size effect-mediated surface oxidation states can directly modulate the antioxidant activity of Ru NPs.

3.2.2 Morphology engineering

Morphology has an important effect on the catalytic activity of nanozymes, including crystallographic surface, surface cut, and surface energy, which affects their enzyme selectivity and reactivity^[29,63]. For example, different morphologies of cerium-based nanomaterials (such as nanoclusters, nanoparticles, and nanoclains) were synthesized. Among them, cerium nanoclusters showed the highest level of SOD-like activity, and cerium nanoclusters showed higher SOD-like activity than CeNPs at cerium concentrations ranging from 5 to 100 mg/L. Similarly, among manganese-based nanozymes of different shapes (such as nanoflowers, plates, cubes, polyhedra, and hexahedra), flower-shaped Mn₃O₄ (with a specific surface area of up to 97.7 m²/g) exhibits the broadest catalytic activity, including CAT, GPx, and SOD-like activity, while other morphologies only exhibit SOD-like activity^[64]. Ghosh et al.^[65] synthesized 4 different morphologies of V₂O₅ NPs, such as nanowires, nanosheets, nanoflowers, and nanorods, and found that the rate of formation of peroxides on the surface affects their GPx-like activity. VO₂ nanofibres were reported to have higher peroxidase-like activity compared with VO₂ nanosheets and VO₂ nanorods. Reported that (111)-faceted Pd octahedra with lower surface energy exhibited higher antioxidant activity than (100)-faceted nanocubes with higher surface energy (Figure 3B)^[66]. Different morphologies of Co₃O₄ nanozymes exhibited different degrees of CAT-like activity in the order of Co₃O₄ nanoplates > Co₃O₄ nanorods > Co₃O₄ nanocubes^[67]. In general, nanozymes with large specific surface area, including flower-like, nanoclusters, nanofibres, and mesopores, tend to exhibit higher enzyme activity.

3.2.3 Composition tuning

The catalytic activity of nanozymes can be modulated by adjusting the composition of the nanomaterials, which provides a variety of design strategies for optimizing the synthesis and performance of nanostructures^[68]. For example, the overall activity can be enhanced by doping with other ions.

It is well known that the Ce³⁺/Ce⁴⁺ ratio affects the ability of CeO₂ NPs to scavenge ROS. Doping is a successful and relatively simple strategy to improve the antioxidant capacity of CeO₂. Researchers have developed CeO₂ nanozymes by doping with different metal elements, including manganese, cobalt, silver, chromium, rhodium, palladium, and nickel. Gupta et al.^[69] successfully synthesized La-, Sm-, and Er-doped CeO₂, which controllably altered the ratio of Ce³⁺/Ce⁴⁺ and significantly improved the antioxidant properties of CeO₂. In the study by Zhang et al.^[70], the addition of Cr³⁺ increased the surface ratio of Ce³⁺/Ce⁴⁺, thus improving the catalytic activity of CeO₂ nanozymes. Sun et al.^[71] synthesized a series of halogen-doped CoCN, obtained CoCNF, CoCNCl, CoCNBr and CoCNI. We found the CAT, SOD, and GPx-like catalytic activities of CoCNCl, CoCNBr, and CoCNI significantly increased than that of CoCN. Especially, CoCNI exhibited the highest antioxidant capacity.

The reasonable design of bimetal or multimetal nanocomposites can help improve the catalytic properties of nanozymes. Wang et al.^[72] reported a 1-step solvothermal synthesis of Au-doped Fe₃O₄ nanoparticles (Au@Fe₃O₄) with much higher catalytic activity than Au and Fe₃O₄ NPs. The catalytic performance and Raman scattering activity of AgAu, AgPd, and AgPt NPs were more pronounced than those of Ag NPs^[73].

Adjusting the ratio of components and designing nanomaterials based on metal core/shell structures are both viable solutions to modulate enzyme-like properties. In general, gold nanoparticles alone do not exhibit SOD-like activity even at very high concentrations (300 µg/mL)^[74]. However, Au core/Ce shell-based nanozymes (Figure 3C) exhibit SOD-like activity over a wide pH range (2–11) and at temperatures up to 90 °C.

3.2.4 Surface modification

Surface modification is another factor that highly influences the antioxidant activity, as most of the antioxidant reactions occur on the surface of nanomaterials. The main substances used for surface modification include polymers, biomolecules, small molecules, and ions, which can be modified to the surface of the material by physical adsorption or covalent coupling. In addition, different types of modifications can form coatings of different thicknesses, functional groups, and surface charges, which can affect the catalytic properties, stability, and substrate specificity of nanomaterials^[68].

Huo et al.^[75] modified Co₃O₄ nanoplates with the amino group (NH₂-Co₃O₄), carboxyl group (COOH-Co₃O₄), hydroxyl group (OH-Co₃O₄), and sulfhydryl group (SH-Co₃O₄), respectively, and then systematically studied their catalytic activities (Figure 3D). Except hydroxyl group, the other functional groups all possessed positive effect to enhance POD-like activities, and among which the NH₂-Co₃O₄ nanoplates ranked the first. The researchers considered that the functional groups’ influence on the electron transfer ability of nanozymes was critical to modulating their catalytic properties^[29].

Ligands such as DNA, GSH, proteins, and dendritic polymers help to prevent the aggregation of metal nanoclusters, which enhances the equilibrium, biocompatibility, and enzyme activity of the nanozymes^[76]. Yang et al.^[77] successfully prepared polyethylene glycol (PEG)-modified single-atom nanozymes (Cu-SAzymes) with divalent Cu-N₄ structure, and the results showed that PEG-modified Cu-SAzymes significantly improved the ability to scavenge intracellular O₂ and effectively reduced oxidative DNA damage. Liu et al.^[78] reported that PAMAM dendrimerised gold nanoclusters (AuNCs-NH₂) terminated with amines unexpectedly lost POD-like activity under physiological conditions, but retained CAT-like activity. This may be due to the enrichment of polymeric tertiary amines, which exerted sufficient inhibitory effect on the key mediator -OH.

Meanwhile, poly tannic acid (PTA)-coated CeO₂ nanoparticles exhibited more prominent SOD-like activity than untreated CeO₂, which was attributed to the fact that the intrinsic reducing property of PTA might contribute to the enhancement of the Ce³⁺/Ce⁴⁺ ratio on the surface of CeO₂, thus promoting the antioxidant activity^[79]. CeO₂ NPs (4.5 nm) were encapsulated into the cavities of the biomolecule apoferritin to form apoferritin-CeO₂ nanoclusters^[80]. The apoferritin coating improved the biocompatibility of the CeO₂ NPs and controlled the electron localization on the surface of the nanomaterials, resulting in enhanced ROS-scavenging activity.

3.2.5 External stimuli

In addition to controlling the above factors, the activity of nanozymes can also be regulated by controlling conditions such as pH, light, and temperature. Several metal- and metal oxide-based nanozymes have been found to have enzyme-like activity in response to changes in pH and temperature^[81]. For example, AuNPs exhibit the catalytic ability of peroxidase in acidic environments, while showing catalytic properties similar to SOD or CAT at alkaline pH^[82]. Pt NPs^[83], Ag NPs^[84] have been reported to act as POD mimics under acidic conditions, while exhibiting CAT-like activity in neutral and alkaline environments. More importantly, Pt and Au NPs were shown to display SOD mimicry under neutral conditions^[85]. Gao et al.^[1] reported the POD-like activity of Fe₃O₄ NPs at pH 3.5. Chen et al.^[86] reported that Fe₃O₄ and γ-Fe₂O₃ NPs exhibited CAT-like activity under neutral conditions (Figure 3E). Wu et al.^[87] synthesized FeCO@C with OXD and POD-like activities. The OXD activity was strong at the pH of 3.6; however, it decreased significantly when the pH was increased to 4.4, while the POD activity increased.

Light is also used as an ideal external stimulus to regulate catalytic reactions. Xu et al.^[88] selected the photothermal material graphene oxide (GO) and the photobase reagent malachite green carbinol base (MGCB) to construct a dual-responsive system (GO-MGCB), which can be temperature- and pH-modulated under UV and NIR irradiation. Studies have shown that temperature has a significant effect on the catalytic activity of copper-loaded tin oxide (SnO₂-Cu) nanocomposites in POD mimicry reactions. The temperature range from 22 to 40 °C is where the color intensity and UV-visible absorption are highest. Beyond 40 °C, the absorbance starts to decrease and is lowest at 80 °C. Surprisingly, the maximum catalytic activity was observed at a modest temperature of 22 °C (Figure 3F). Varying the reaction parameters of the SnO₂-Cu nanocomposites is important for this temperature-dependent behavior^[89].

Photothermal effects and light-induced electron transfer have been shown to be the main sources of light-enhanced enzyme-like behavior of nanozymes^[29]. For example, Researchers used AuNPs with α-FeOOH microcrystals formed on porous carbon to develop Au/α-FeOOH-FPC catalysts with visible-light-driven enzymatic properties^[90]. By absorbing light energy, the AuNPs can emit heat, thereby increasing the system temperature to the desired level for the enzymatic process and accelerating the glucose oxidation reaction. In addition, hot electrons from the equipartitionally excited AuNPs promote charge separation at the Au/α-FeOOH interface, leading to the efficient cycling of Fe³⁺/Fe²⁺ to generate the Fenton reaction.

4.1.1 Metal nanozymes for GI

Metal-based nanozymes possess several advantages of easy preparation, special optical properties, excellent chemical stability under extreme conditions, and high biocompatibility^{[96–97–98]}. As shown in Table 1, these nanozymes mimic natural enzymes by initiating and facilitating specific reactions and are capable of stable enzyme-like activity under physiological conditions^[2]. Studies have shown that bismuth selenide 2-dimensional nanodisks (Bi₂Se₃) not only possess the ability to scavenge ROS and RNS but also regulate intestinal microbiota balance, increasing the abundance of Firmicutes and Actinobacteria while reducing the abundance of pathogenic Actinobacteria, thereby maintaining intestinal microbiota balance^[99]. Halogen-doped modified single-atom cobalt catalysts (CoCN) exhibit exceptional antioxidant enzyme activity, significantly improving symptoms of colitis^[71]. Zero-valent molybdenum nanoparticles, with their strong reducing capacity and acid stability, can effectively downregulate the NF-κB pathway, inhibit the overproduction of inflammatory factors, and alleviate intestinal oxidative damage^[100]. Molybdenum selenide (Mo₃Se₄) nanosheets (PMNFs) exhibit multiple enzymatic catalytic activities, alleviating oxidative damage through the Nrf2-Keap1 signaling pathway while inhibiting the TLR4/NF-κB signaling pathway to reduce proinflammatory factor levels, thereby preventing damage to intestinal epithelial TJ proteins (ZO-1, occludin, and claudin-1), while downregulating Dextran Sulfate Sodium (DSS)-induced intestinal epithelial cell apoptosis^[102]. Ultrafine rhodium nanoparticles (Rh-PEG NDs) possess multienzyme mimetic activity (eg, CAT) and high photothermal conversion efficiency, enabling them to scavenge ROS and RNS for the treatment of DSS-induced colitis and serve as photothermal agents for photothermal therapy of colorectal cancer^[101].

Notably, tungsten diselenide nanocatalysts (WSe₂@F127) achieve reactive oxygen/nitrogen species (RONS) scavenging through a rich valence state conversion mechanism involving elemental selenium (Se²⁺/Se⁴⁺) and tungsten (W⁴⁺/W⁶⁺), and specifically regulate intestinal microbiota and immune barriers via tungsten-mediated inhibition of Enterobacteriaceae and selenium-mediated T-cell differentiation^[103]. Meanwhile, ternary metal nanosheets (Ru₃₈Pd₃₄Ni₂₈, abbreviated as TMNSs) seamlessly integrate antioxidant therapy with photothermal therapy. By introducing different transition metal atoms into the ruthenium-palladium nanoplate structure, the formation of Ru-O and Ni-O bonds on the TMNS surface is promoted, and the abundant atomic vacancies in its surface design significantly enhance the clearance performance of ROS. TMNS can downregulate the expression levels of proinflammatory factors, thereby demonstrating significant therapeutic effects on DSS-induced colitis. Leveraging its excellent photothermal performance, TMNS can significantly inhibit CT-26 tumors without noticeable recurrence^[108].

Some studies have shown that coordination polymer nanozymes based on natural antioxidant products and metal ions have promising applications in clinical translation for the treatment of GI. For example, proanthocyanidins (Pc) and free iron (Fe) coordinate to form a stable Pc-Fe nanozyme that mimics the activity of POD and GPx, exhibiting excellent antioxidant capacity and ROS-scavenging ability in vitro. Further in vivo studies revealed that inflammation regulation and gut microbiota regulation may be the potential mechanisms underlying the good therapeutic effects of the Pc-Fe nanozyme on the DSS-induced colitis model, particularly in terms of downregulating proinflammatory cytokines, reducing inflammatory cell infiltration, and restoring gut microbiota dysbiosis^[105]. Another innovative research and development project involved ultrasmall tungsten-gallic acid (W-GA) nanodots, which precisely regulate the intestinal microbiota by inhibiting the abnormal proliferation of Enterobacteriaceae bacteria during colitis, thereby promoting the restoration of the intestinal barrier. These nanodots integrate microbiota reprogramming and mitigate the damage caused by oxidative stress to the reconstruction of bacterial communities, demonstrating good therapeutic effects on IBD^[104].

Taxifolin-iron nanozymes (Fe-Tax) effectively remove reactive oxygen and nitrogen species in the gastrointestinal tract by mimicking SOD and CAT, thereby alleviating oxidative damage, inflammatory responses, and cell apoptosis. Additionally, Fe-Tax could alleviate tissue inflammation and gastric mucosal damage by regulating NRF2, NF-κB, Bax/Bcl-2, and VEGF signal pathways in ethanol-induced gastric ulcer^[111]. Using fully edible natural active polyphenols, iron ions, and polyvinylpyrrolidone, sub-10 nm ultrasmall iron-polyphenol nanozymes (UIPNs) were synthesized (Figure 5A). UIPNs rapidly penetrate the gastric mucosa, remain in the stomach for over 12 hours, and maintain stability in a strongly acidic environment. They exhibit outstanding enzymatic activity for CAT, POD, and SOD, and demonstrate excellent performance in preventing and protecting against alcohol-induced gastric mucosal damage in mice^[112]. These studies collectively demonstrate that the synergistic coordination of polyphenols (such as proanthocyanidins and quercetin) with metal ions (Fe/Cu/Mn, etc) not only preserves the high catalytic activity of metal nanozymes but also reduces the toxicity of metal ions through natural antioxidant ligands, thereby enhancing their biocompatibility. This provides an innovative solution for the clinical treatment of GI disorders.

4.1.2 Metal oxide nanozymes for GI

4.1.3 Metal-organic frameworks for IBD

MOFs are porous hybrid materials composed of metal ions/metal clusters and organic ligands. The excellent properties of MOFs, such as structural tunability, functional group richness, large specific surface area, and high thermal stability, have driven the research on their applications in biomedical fields^[2,73,134]. In GI treatment applications, MOF materials mainly exert their effects through 2 strategies. The first is as nanocatalysts with intrinsic enzyme activity, and the second is as multifunctional carriers for delivering therapeutic drugs or active substances (Table 3).

By carefully selecting metal centers and organic ligands, MOF materials with specific enzyme-mimicking activity can be constructed. For example, functionalization of ligands in the MIL-47(V) series of MOFs by introducing different substituents (such as -NH₂, -OH) can significantly regulate their GPx-like activity, with the amino-modified MIL-47(V)-NH₂ exhibiting excellent biocompatibility and ROS-scavenging ability^[135]. More complex systems, such as the PCN222-Mn/Pt composite material constructed by combining Mn(III) porphyrin ligands with Pt nanoparticles, achieve cascading catalytic effects of SOD and CAT, efficiently converting ${\mathrm{O}}_2^{-}$ into harmless water and oxygen, and effectively protecting mice from ROS-related IBD^[136]. Additionally, ruthenium-based MOFs (Ru-MOFs) integrate the catalytic properties of tris(phenylphosphine)ruthenium dichloride, demonstrating broad-spectrum ROS-scavenging capacity and stable multienzyme activity, showing significant therapeutic effects in DSS-induced colitis models^[137]. Addressing the challenge of H pylori gastritis infection, the innovative Cu-MOF@NF platform is loaded with nitrogen-doped carbon dots and fucoidan (Figure 5B). Not only can it penetrate the gastric mucus layer and prevent H pylori from adhering to gastric mucosal epithelial cells, but the released Cu²⁺ can also effectively degrade biofilm polysaccharides and disrupt the cyclical growth pattern of “bacterial plankton-biofilm,” thereby preventing recurrent and persistent infections^[148].

Prussian blue (PB) and its analogues are a common class of MOFs with antioxidant enzyme activity, consisting of trivalent iron and ferrous iron coordinated with cyanide, which are biocompatible and biosafe. PB NPs have catalytic properties similar to those of POD-, CAT, and SOD, which may be attributed to interconversions between the different valence states of Fe, which make PB NPs good electron transporters^[149]. Based on the PB framework, researchers have developed various functionalized nanozyme systems. Manganese-doped PB nanoparticles (MPBs) not only retain the multienzymatic activity of PB but also serve as drug carriers for loading the anti-inflammatory drug sulfasalazine. This nanomaterial system improves cellular energy dysfunction, inhibits apoptosis, and exhibits strong anti-inflammatory and immune-balancing capabilities in IBD^[139]. Selenium-doped PB (Se-HMPB) introduces selenium with GPx-like activity, effectively inhibiting iron-dependent cell death in UC treatment and regulating Th1 cell differentiation in CD treatment, thereby effectively protecting the integrity of the intestinal mucosal barrier^[143]. Notably, the ZnPBA@YCW oral delivery system, formed by PB analogues using yeast cell walls as carriers, specifically targets pathogenic Escherichia coli, eliminating pathogens while regulating intestinal microbiota balance. It also reduces inflammatory responses by inhibiting the NF-κB signaling pathway, achieving synergistic effects through multiple therapeutic mechanisms^[144].

In addition, MOF, as a framework material, can deliver nanozymes or actives for site-specific treatment of GI. By co-encapsulating carbon dots (named as CNs) with SOD activity and CD98 CRISPR/Cas9 plasmids (named as CPs) within ZIF-8nanoparticles (termed as CCZ) enveloped by macrophage membranes, the constructed CCZM system exhibits acid-responsive release, immune evasion, and inflammation-targeting properties. It effectively clears ROS at the lesion site and downregulates the expression of the inflammation-related biomarker CD98, significantly improving inflammatory symptoms in mice, including colon length, epithelial integrity, and inflammatory response, thereby achieving a synergistic therapeutic effect (Figure 5D)^[145]. Similarly, the CCM-Co-ZIF-8 composite material formed by cobalt-doped ZIF-8 loaded with curcumin not only possesses multiple ROS-clearing enzyme activities (POD, CAT, and SOD) but also promotes the polarization of macrophages from proinflammatory M1 type to anti-inflammatory M2 type, effectively regulating the immune microenvironment^[146]. Cobalt-doped ZIF-8 loaded with tannic acid forms tannic acid-zinc nanoparticles (HZn-TA), which alleviate intestinal inflammation in mice with acute colitis by reducing colonic damage, proinflammatory cytokine levels, spleen index, and body weight. It also promotes intestinal mucosal healing by upregulating the expression of TJ proteins ZO-1 and claudin-1^[147].

4.2.1 Carbon-based nanozymes for GI

Carbon-based materials are rich in surface functional groups and have many unsaturated structures, leading to a large number of active sites and multiple enzyme activities^[26,150]. Carbon-based nanozymes are highly biocompatible, relatively stable in living organisms, easy to be functionally modified, and widely used in GI therapy (Table 4).

In UC and CD enteritis models, carbon dots (C-dots) synthesized from glutathione and biotin exhibit significant SOD-like activity, effectively scavenging ROS and reducing the expression of proinflammatory factors such as TNF-α and IL-6, thereby effectively repairing damaged intestinal mucosa^[151]. C-dots encapsulated in chitosan/alginate hydrogels not only possess anti-inflammatory and antioxidant functions but also regulate intestinal microbiota balance, increase the abundance of probiotics, and reduce the number of pathogenic bacteria. Additionally, their red fluorescence properties can be used for bioimaging of inflammatory sites (Figure 6A)^[152]. Metal-free carbon dots (CP-CDs) prepared from citric acid and polyethylene polyamine exert their effects through a dual mechanism. On the one hand, they alleviate inflammatory responses by regulating oxidative stress and inhibiting the NLRP3 inflammatory pathway. On the other hand, they restore the balance of the intestinal microbiota, increase the abundance of beneficial bacteria (Ligilactobacillus and Enterorhabduus), reduce the abundance of harmful bacteria (unclassified Clostridia UCG-014), and promote the recovery of intestinal barrier function^[153].

In the field of H pylori infection treatment, pH-responsive fullerene alcohol nanoparticles (FNPs) have achieved breakthrough progress^[157]. FNPs achieve precise antibacterial effects through molecular structural transformations triggered by gastric acid. In an acidic environment, the hydroxyl groups of FNPs rearrange to form active carbonyl groups (C=O), significantly enhancing their POD activity, with catalytic efficiency positively correlated with C=O content. These nanoparticles can specifically destroy the cell walls and biofilms of H pylori, and are equally effective against drug-resistant strains. Experimental evidence shows that they function through a reversible substrate binding-catalytic cycle, offering advantages such as high selectivity and no development of drug resistance, thereby providing an innovative nonantibiotic solution for H pylori treatment.

Metal-doped carbon-based nanozymes have demonstrated breakthrough progress in the treatment of GI through multienzyme synergistic action and intelligent response design. Iron/nitrogen codoped hollow porous carbon nanospheres (Fe/N-HCNs) exhibit 4 enzyme-mimicking activities: POD, OXD, CAT, and SOD. These not only effectively scavenge ROS but also regulate the inflammatory microenvironment through enzymatic reactions, demonstrating significant therapeutic effects in bacterial infection and DSS-induced colitis models^[156]. Similarly, platinum-doped carbon nanodots achieve efficient ROS scavenging by mimicking the SOD/CAT cascade reaction. When coloaded with spleen tyrosine kinase inhibitors in platelet membrane-coated nanoparticles, they can precisely target inflammatory sites, inhibit neutrophil infiltration, and promote epithelial repair, thereby reversing intestinal barrier dysfunction^[160]. To further optimize the stability and catalytic efficiency of nanozymes, researchers developed iodine-copper-zinc covalently doped carbon dots (Cu, Zn, I-CDs), where iodine doping effectively neutralizes the positive charge of metal ions in acidic environments, enabling them to maintain excellent triple antioxidant activity (CAT, SOD, and GPx) even under low pH conditions. In vitro experiments confirmed that this material not only alleviates cellular oxidative damage but also downregulates the expression of proinflammatory factors such as TNF-α and IL-1β. In a DSS-induced colitis model, it significantly improves pathological features such as colon shortening and mucosal damage (Figure 6B)^[154]. For H pylori infection, pH-responsive nanozymes (such as PtCo@G) achieve selective bactericidal activity in the gastric acid environment through surface modification with phenylboronic acid. Their catalytic activity is activated only under acidic conditions, thereby avoiding unintended damage to the intestinal microbiota^[158]. Additionally, dual-targeted nanozymes (PtCo@G@H2A) enhance targeting efficiency by 850% through a dual mechanism of heme receptor binding and pH-dependent charge reversal (L-arginine protonation). They also generate selective ROS via cascade catalytic reactions, maintaining high bactericidal efficacy in gastric acid and pepsin environments while avoiding intestinal side effects through surface potential regulation^[159].

4.2.2 Melanin nanozymes for GI

Metal-free melanin nanomaterials can also be effective in the treatment of GI, as summarized in Table 5. Melanin-based nanomaterials (MeNPs) utilize their unique phenol/quinone redox structure to efficiently scavenge ROS, demonstrating multitarget regulatory capabilities in IBD treatment. They can simultaneously alleviate 6 major pathological features: oxidative stress, endoplasmic reticulum stress, cell apoptosis, inflammatory response, intestinal epithelial damage, and gut microbiota dysbiosis^[161]. To further enhance targeting and therapeutic efficacy, researchers developed a probiotic-nanocatalyst composite system (Lf@MPB) by modifying melanin nanoparticles onto the surface of Lactobacillus fermentum. This not only enhances the survival rate of probiotics in the gastrointestinal environment but also achieves precise accumulation at inflammatory sites. Experiments confirmed that this system significantly reduces mitochondrial membrane potential, inhibits cell apoptosis, and exerts synergistic therapeutic effects by regulating the composition of the intestinal microbiota^[162]. Additionally, allomelanin-like nanodrugs (alloMel) demonstrate superior performance, with antioxidant capacity 2.5-fold that of conventional drugs such as 5-aminosalicylic acid. They specifically target colitis lesions by inhibiting the TLR4/MyD88/NF-κB signaling pathway, effectively regulating macrophage polarization, reducing inflammatory cell infiltration, and restoring intestinal barrier integrity (Figure 6C)^[163]. These melanin-based nanocatalysts offer a novel metal-free nanocatalytic therapeutic strategy for GI diseases.