Natural enzymes are important functional substances in organisms. They also play an indispensable role in genesis and survival of tumor [1-3]. Metabolic changes caused by natural enzymes during tumor development cannot be ignored, such as large production of proteins [4], lipids [5] and nucleic acids [6] as well as consumption of energy substances such as carbohydrates [2,7]. Therefore, targeting natural enzymes is an effective strategy for tumor treatment [8,9]. Factors such as substrate and enzyme concentration during enzyme reaction will promote tumor cell death [10,11]. However, the levels of natural enzymes in organisms are extremely low, the influencing factors are difficult to regulate [12], and the synthesis of natural enzymes is limited by complex spatial structures, cumbersome synthesis processes, expensive production costs, and specific storage conditions [13,14]. Hence, nanozymes have been developed to overcome these difficulties and meet the needs of tumor therapy.

Nanozymes are synthesized artificially and possess the properties of nanomaterials and catalytic functions of enzymes [15]. Compared with natural enzymes, nanozymes have simpler structures, easier preparation and easier storage. Under physiological conditions, nanozymes follow the same kinetic mechanism as natural enzymes and can effectively catalyze the conversion of substrates. Surprisingly, some nanozymes with specific structures can exert multiple enzymatic activities. Their excellent catalytic efficiency and biocompatibility open a new field for applying nanozymes in tumor therapy [16,17]. In tumor sites, nanozymes use metabolites in the tumor microenvironment as substrates and catalyze the generation of active substances with properties that lead to cell death [18]. In addition, nanozymes can be used as carriers to deliver conventional drugs to the tumor site. In combination with the properties of the nanomaterials themselves, nanocatalytic therapies are also often combined with other therapies to produce detrimental effects to tumor cells, such as photothermal therapy (PTT), sonodynamic therapy (SDT), photodynamic therapy (PDT), and chemical-dynamic therapy (CDT). Despite the excellent catalytic properties of nanozymes, the lack of reaction substrates in the tumor microenvironment and the limitation of reaction conditions prevent the advantages of nanozymes from being fully exploited [19]. Therefore, there is an urgent need to develop nanozyme systems with reaction substrates and conditions.

Here, a nanozyme (HAuHbO₂) consists of hollow gold nanorods (HAuNPs) loaded with oxygen-carrying hemoglobin (HbO₂) was designed to achieve antitumor therapy by spontaneous cascade catalytic function under near-infrared (NIR) radiation (Scheme 1). The glucose oxidase (GOD)-like and peroxidase (POD)-like activities of HAuHbO₂ can effectively catalyze the production of cytotoxic ^•OH in the tumor microenvironment and stimulate tumor cell death. The anti-tumor experiments results indicate that HAuHbO₂ is an effective therapeutic reagent. More importantly, HAuHbO₂ have good biosafety. In summary, the study could provide a new understanding of the role of nanozymes in tumor therapy (Table S1 in Supporting information).

HAuHbO₂ is prepared by binding sulfhydryl groups on HAuNPs and Hb to form disulfide bonds. The stability of HAuNPs and the biological activity of Hb were both well preserved, which was confirmed by the following experiments. First, transmission electron microscopy (TEM) was used to demonstrate the morphological features of the nanozyme. The rod-like morphology of TeSe, HAuNPs and HAuHbO₂ can be visualized clearly in Fig. 1a. The hollow structure of HAuNPs increases the surface area of the material, making it an effective carrier. A thin film can be visualized on the surface of HAuHbO₂ demonstrating the successful loading Hb onto the HAuNPs. Additionally, elemental mapping further analyzes the composition of HAuHbO₂ at Fig. 1b. The characteristic absorption peaks of Hb, HAuNPs, and HAuHbO₂ were measured by ultraviolet-visible (UV–vis) spectrophotometer (Fig. 1c). HAuHbO₂ was found to have characteristic peaks of Hb and HAuNPs, indicating the successful construction of the nanozyme. The hydrodynamic sizes of HAuNPs and HAuHbO₂ are shown in Fig. 1d, which are 125 and 170 nm, respectively. It indicates that the particle size is increasing from HAuNPs to HAuHbO₂. In addition, it was determined in Fig. 1e that the zeta potentials of Hb, HAuNPs, and HAuHbO₂ were −1.06, −5.96, and −2.48 mV, respectively. These experimental results confirmed the successful preparation of the nanozyme and revealed the potential functions, including PTT and photoacoustic (PA) imaging.

In order to demonstrate the potential of HAuHbO₂ as a PTT agent, the photothermal effect was investigated by monitoring the thermal activities with 808 nm (1.0 W/cm²) continuous laser irradiation. As shown in Fig. 1f, phosphate buffered saline (PBS) showed the lowest response to laser. Notably, Hb exhibited photothermal properties under the same experimental conditions. It is consistent with the conclusions of previous studies that Hb has photothermal conversion ability [20,21]. Therefore, the final temperature of HAuHbO₂ was higher than HAuNPs. The photothermal images of the four samples after laser are shown in Fig. 1f. In addition, when subjected to five heating/cooling cycles, the temperature change of HAuHbO₂ remained stable (Fig. 1g). It demonstrated the good photothermal stability of HAuHbO₂. The photothermal conversion efficiency (η) was calculated to be as high as 31.09% under 808 nm irradiation (Fig. 1h). The good photothermal stability and conversion efficiency of HAuHbO₂ make it a potential PTT for tumor therapy.

As Hb has the function of oxygen carrier, its oxygen carrying capacity was also verified here. As shown in Fig. S1 (Supporting information), oxygen content in deoxy-PBS, HAuHb and HAuHbO₂ solutions was measured. The result shows that HAuHbO₂ had the highest dissolved oxygen, indicating its oxygen carrying capacity. In addition, the oxygen carrying stability of HAuHbO₂ was also evaluated. As shown in Fig. 1i, deoxy-PBS simulates an anoxic tumor microenvironment, releasing oxygen rapidly after nanozyme enters the solution, and then maintaining a stable oxygen release rate. This experiment provides evidence for the function of nanozyme in alleviating hypoxia in tumor therapy. The hydrodynamic diameter of HAuHbO₂ remained stable without any substantial change in water, PBS, and DMEM for 7 days (Fig. S2 in Supporting information), which indicated its good stability.

The enzyme mimetic activities of the nanozymes were evaluated using the electron paramagnetic resonance (EPR) technique. First, the generation of H₂O₂ was verified. In the system of glucose mixed with nanozymes, the generated superoxide hydroxyl radical (^•O₂⁻) gradually evolved into superoxide hydroxyl radical (^•OOH) as the reaction proceeded and was rapidly converted to H₂O₂ by protonation [22]. The EPR system focused on the intermediate reactive species ^•O₂⁻ in the pathway of H₂O₂ generation. Fig. 1j demonstrates the ability of HAuNPs, HAuHb, and HAuHbO₂ to generate ^•O₂⁻. Three samples showed the possibility that H₂O₂ was generated. It is noteworthy that the strongest signal was found in the HAuNPs, which may be the H₂O₂ in HAuHb and HAuHbO₂ had already reacted in the later step. After subjecting the reaction system to laser irradiation, the catalytic efficiency of HAuNPs was found to be significantly improved (Fig. S3 in Supporting information), whereas no signals were detected in the HAuHb and HAuHbO₂, which inferred that the generated H₂O₂ had already been engaged in the subsequent reaction. The occurrence of the second-step reaction was also investigated. As shown in Fig. 1k, the nanozymes exhibited significant ^•OH generation. The characteristic signal of ^•OH appeared in the glucose and nanozyme mixed system. It was not detected in the component HAuNPs system alone, implying the lack of POD-like activity without Hb. Similarly, the signal enhancement upon laser irradiation indicates that light can facilitate the reaction (Fig. S4 in Supporting information). Compared with HAuHb and HAuHbO₂, adding more oxygen was seen to promote the reaction. In conclusion, these results strongly demonstrate the enzyme catalytic activities of the nanozyme with GOD and POD, which further extend the therapeutic potentials for tumor treatment.

Different experimental approaches were further used to evaluate enzyme mimetic properties. HAuNPs catalyze glucose and O₂ into gluconic acid and H₂O₂ (Fig. 2a). H₂O₂ generation was detected using indigo carmine (IC) as an indicator. As shown in Fig. 2b, the absorbance of IC was the highest but was the lowest in HAuNPs+IC. It indicated that HAuNPs catalyzed the generation of H₂O₂. Meanwhile, the absorbance of HAuHbO₂+IC was also reduced but larger than HAuNPs+IC. It is caused by the Fenton reaction, which consumed H₂O₂ in the system. The color changes of IC can be seen in the photo at the upper right of the UV map. Subsequently, the kinetics of the GOD-like reaction of HAuNPs was investigated (Fig. 2c). It was shown that the K_m of HAuNPs was 5.972 mmol/L, and the catalytic efficiency (kcat/km) could reach 849.4 L mol⁻¹ s⁻¹.

The mechanism of POD-like activity was subsequently revealed. 3,3′, 5,5′-Tetramethylbenzidine (TMB) is a commonly used probe for ·OH detection (Fig. 2d). Fig. 2e shows no significant absorption was observed in TMB, TMB+Glu and HAuHbO₂. However, a clear absorption peak at 652 nm was observed in TMB+Glu+HAuHbO₂, which indicating that the production of ^•OH. This difference is also illustrated by the apparent change in color between solutions. The effects of glucose and HAuHbO₂ concentrations on the cascade reactions were also verified (Figs. 2f and g). Fig. 2h shows the effect of laser irradiation to catalytic reaction, which is consistent with the results of EPR. The POD-like reaction kinetics of HAuHbO₂ were also investigated (Fig. 2i). The results showed the catalytic kinetics of HAuHbO₂ (K_m = 51.36 mmol/L, V_max = 1.76 × 10⁻⁶ mol L⁻¹ s⁻¹).

Finally, the mechanism of the GOD-like catalytic activity was explored by density functional theory (DFT). When glucose is not added by a catalyst to produce H₂O₂, external energy must be added (Fig. S5 in Supporting information), which limits the production of H₂O₂ in the natural state. Two nanoclusters, Au(Ⅲ) and Au₃₈, were considered for the study of the catalytic interface (Fig. S6 in Supporting information). In the reaction system, glucose molecules and O₂ are first adsorbed on the gold nanoclusters, and then -Hs on the glucose molecules are transferred to O₂ to finally form H₂O₂. The energy changes during this reaction are shown in Fig. 2j. When Au(Ⅲ) was used as the catalytic surface, O₂ was dispersed near the glucose, and then two O atoms of glucose and O₂ were bound to the Au surface sites, respectively. Then, -Hs on glucose are transferred to O₂ to generate the complex C₆H₁₁O₆*_OOH* (* indicates the adsorption state). -OOH is favorable on the Au surface and can break the two hydrogen bonds between -OOH and C₆H₁₁O₆ by rotating -OOH*. The process absorbs heat and transfers it. Upon -OOH transfer, another H atom on the glucose molecule spontaneously dissociates and moves toward the -OOH to form H₂O₂ (Fig. S7 in Supporting information). Overall, the process that produces H₂O₂ is kinetically and thermodynamically favorable, with the rate-limiting step forming the C₆H₁₁O₆OOH complex. In contrast, the catalytic process on the Au₃₈ surface requires less energy. Unlike Au(Ⅲ), the adsorption of glucose onto the Au₃₈ surface requires energy absorption from the outside. Moreover, only one O atom in O₂ is adsorbed on the Au₃₈ surface (Fig. S8 in Supporting information). Compared with Au(Ⅲ), a smaller energy change occurs in the process of -OOH breaking during Au₃₈ catalysis. Therefore, it is conceivable that Au₃₈ exhibits a faster reaction rate. These data indicate that HAuHbO₂ has an excellent enzymatic activity, which will provide a basis for its remarkable therapeutic effect in tumor microenvironment (Fig. 2k).

Further, antitumor effects of HAuHbO₂ were explored in 4T1 tumor cells. The nanozyme labeled by the fluorescent dye Cy5.5 (Cy5.5+HAuHbO₂) were shown to accumulate over time in the cells (Fig. 3a). Flow cytometry also showed consistent accumulation of Cy5.5+HAuHbO₂ in cells (Fig. 3b). The localization of the nanozyme in the cells was captured by bio-electron microscopy (Fig. 3c). All experimental results confirmed that HAuHbO₂ could be effectively phagocytosed by the cells. The cytotoxicity of HAuHbO₂ was further investigated. As shown in Fig. S9 (Supporting information), HAuNPs maintained a survival rate of > 80% at concentrations of 0–0.48 mg/mL, indicating that the nanozyme has almost no harmful effects on tumor cells with good biosafety. HAuHb and HAuHbO₂ showed destructive effects on tumor cells, possibly caused by the spontaneous cascade catalytic reaction. The nanozyme demonstrated excellent antitumor effects after laser irradiation (Fig. 3d). In order to prove the synergistic ability of catalytic therapy and PTT, the combination index (CI) was calculated. The results showed a CI of 0.822 indicating synergistic efficacy of nanozyme. Calcein-AM/PI staining studies demonstrated a direct visualization of 4T1 cells in each group. The strongest red fluorescence of HAuHbO₂ after laser can be seen in Fig. S10 (Supporting information). Apoptosis of cells in each group was detected by flow cytometry using Annexin V-FITC/PI kit. HAuHbO₂+NIR group having the highest mortality rate of 37.2%. The results of the three assays consistently verified the biosafety and effective anti-tumor results of HAuHbO₂. Finally, 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used to track ^•OH in tumor cells. The green fluorescence signals of HAuNPs, HAuHb, and HAuHbO₂ groups were gradually enhanced (Figs. 3f and g), indicating the production of ^•OH in tumor cells.

Considering the above superior spontaneous cascade catalytic properties and antitumor effects of HAuHbO₂, its therapeutic properties in vivo were investigated. All animal studies were approved by the Ethics Committee of the Laboratory Animal Center of Guangdong Medical University (No. GDMU-2023–00, 010) and followed the guidelines for using laboratory animals in Guangdong Province. Firstly, the biosafety of HAuHbO₂ was investigated using healthy mouse blood. As illustrated in Fig. S11 (Supporting information), no apparent hemolysis was induced and the hemolysis rates among these groups incubated with HAuHbO₂ at various concentrations were all below the standard level of 5%, suggesting favorable hemocompatibility of nanozyme. Then, the biodistribution of HAuHbO₂ was further performed. As shown in Fig. S12 (Supporting information), the fluorescence signals of HAuHbO₂ at the tumor sites were significantly higher than in the free Cy5.5 group at each time point (Figs. S12a and b). The fluorescence of free Cy5.5 group was rapidly metabolized after 8 h of injection. In contrast, the nanozyme group maintained the fluorescence signal longer, indicating that HAuHbO₂ has a strong accumulation capacity at the tumor site. Fig. S12c demonstrates the fluorescence imaging of tumor tissues and major organs obtained from the mice at 24 h. The figure clearly shows that the fluorescence of free Cy5.5 group is not obvious, while the fluorescence of the HAuHbO₂ group is visible. It further shows that the nanozyme have good accumulation in mice. A consistent conclusion can also be obtained from the quantitative fluorescence analysis of Fig. S12d. Based on the absorption properties of HAuHbO₂ in the NIR Ⅱ region, PA imaging was also used to verify the accumulation of HAuHbO₂ at mouse tumor sites. There was no obvious photoacoustic signal in the tumor region before HAuHbO₂ was injected (Fig. S12e). After 1 h of injection, the PA signal gradually enhanced and reached the highest value at the 8th hour (Fig. S12f). At 24 h after injection, the PA signal decreased rapidly and became undetectable. These experimental results demonstrated the ability of the HAuHbO₂ to accumulate at the tumor site. They enabled the determination of the occurrence of PTT at the appropriate time point, which is 8 h after injection.

In addition, the PA imaging system (850 nm) detected the oxygenated Hb (HbO₂) in the tumor vasculature during the accumulation of nanozyme. Fig. S12g demonstrates the signals of HbO₂ in the tumor site at 0 and 8 h after HAuHb and HAuHbO₂ were injected into mice, respectively. It was found that the PA signal in the HAuHb group did not enhance with time. On the contrary, the signal of HbO₂ in the HAuHbO₂ group was similar to the accumulation of nanozyme, and the PA signal reached a maximum after 8 h. It was also illustrated by the quantitative analysis of the HbO₂ signal (Fig. S12h). Therefore, it was concluded that HAuHbO₂ can carry O₂ into the tumor site, which allows spontaneous cascade reaction and relief of tumor hypoxia at the tumor site.

According to the HAuHbO₂ accumulation in mice, the antitumor effects were further explored (Fig. 4a). An infrared thermography camera was used to record the temperature of tumor site and take thermography photos (Fig. 4b and Fig. S13 in Supporting information). The tumor sites of the mice in the PBS group did not show significant temperature changes (temperature change of 3.5 ℃). In contrast, HAuNPs group and HAuHbO₂ group were heated significantly: the temperatures could reach 55.9 and 57.3 ℃. It demonstrated the safe radiation of 808 nm laser and the better photothermal transduction ability of nanozyme, making it sufficient proof for happening of PTT. After, tumor volume and mouse weight were recorded. In Fig. 4c, the tumor volume of the mice in the PBS group showed a significant trend of increase. In contrast, the tumor growth in the HAuHbO₂ group was moderate, which likely attributed to the induction of CDT by the spontaneous cascade reaction of nanozyme. HAuHb+NIR and HAuHbO₂+NIR groups significantly inhibited tumor growth after treatment, and the HAuHbO₂+NIR group could even decreasing tumor volume. It indicates that the synergy of HAuHbO₂ with PTT and CDT in vivo could increase therapeutic effects. In Fig. 4d, the body weight of the mice in all groups showed an increasing trend within 16 days of treatment, indicating that the mice were growing normally, and the nanozyme had a reasonable level of biosafety. The serum, tumor tissues, and major organs were collected after 16 days of treatments. Fig. 4e and Fig. S14 (Supporting information) showed tumor weight and the excised tumors photo, from which the tumor inhibitory effects of the formulations in each group could be visualized. The tumor growth inhibition rate also demonstrated the antitumor ability of the nanozyme (Fig. 4f). HAuHbO₂ was not only catalyzed by cascade to undergo Fenton reaction but also achieved 91.22% tumor inhibition under light-induced thermotherapy. Individual tumor growth curves revealed that tumors in the HAuHbO₂+NIR group may be completely eliminated (Fig. 4g). The effects of the nanozyme on tumor cell proliferation, apoptosis, and necrosis were further evaluated by histological analysis and immunofluorescence staining. In hematoxylin-eosin (H & E) staining of Fig. 4h, the cells in the tumor tissues with HAuHbO₂+NIR treatment showed deformation, nuclear shrinkage, and necrosis, while the morphology of the PBS group remained normal. The results of terminal-deoxynucleotidyl transferase mediated nick-end labeling (TUNEL) staining showed the strong green fluorescence in HAuHbO₂+NIR group, indicating the highest degree of apoptosis. Ki67, as a cell proliferation marker, was used to detect the proliferative ability of tumor cells. It was found that PBS group had obvious fluorescence signals. In contrast, the fluorescence intensity decreased rapidly after nanozyme treatment, indicating that nanozyme inhibited tumor cell proliferation (Fig. 4i) and increased apoptosis (Fig. 4j). Caspase-3 and Bcl-2 are important indicators of apoptosis, so the expression of these two proteins in tumor tissue was also evaluated. As shown in Fig. S15 (Supporting information), caspase-3 in tumor tissue was the highest in HAuHbO₂ + NIR group (Fig. S15a), indicating the possibility of subsequent induction of apoptosis. Bcl-2 as an anti-apoptotic protein, its expression can be seen in the figure to be the lowest in the final group. The results of immunofluorescence quantitative analysis show that nanozyme has a significant therapeutic effect on tumor (Figs. S15b and c).

Subsequently, the biosafety of HAuHbO₂ was verified in vivo. The biosafety of nanozymes can be inferred from Fig. S16 (Supporting information) that no significant damage was observed in the organs of the nanozyme groups compared to the control group. The nanozymes showed non-toxic effects on normal tissues while causing tumor cell death. The liver and kidney function indicators in the serum of the nanozyme groups were also not found to be significantly changed compared with the PBS group (Figs. 4k and l), confirming the biosafety of the nanozymes.

The antitumor mechanism of HAuHbO₂ was further explored. The studies and conclusions of previous studies provide potential mechanisms for the antitumor effects of HAuHbO₂ [23,24]. Firstly, the generation of ^•OH by nanozymes was confirmed in vivo. DCFH-DA was used to detect ^•OH at the tumor site. As shown in Fig. S17 (Supporting information), no green fluorescence was observed in the PBS group, while HAuHbO₂+NIR showed a clear fluorescence signal (Fig. S17a). This confirmation of ·OH generation would provide the possibility that HAuHbO₂ induces oxidative stress to cause immunogenic cell death (ICD). Subsequently, the occurrence of ICD was verified. Calreticulin (CRT) exposure and high - mobility group box 1 (HMGB1) exocytosis is important indicators for ICD development. The expression levels of CRT and HMGB1 in tumor tissues analyzed using immunofluorescence are shown in Fig. S17b. The fluorescence intensity of CRT with the HAuHbO₂+NIR treatment showed the highest level. Meanwhile, the fluorescence of HMGB1 in the PBS group was concentrated in the nucleus and had the highest fluorescence intensity. The expression of HMGB1 was weakened in the other groups as the drug's efficacy was enhanced. Both CRT exposure and HMGB1 exocytosis results demonstrated the occurrence of ICD. Then, the stimulation of dendritic cell (DC) maturation by damage-associated molecular patterns (DAMPs) was analyzed using flow cytometry [25,26]. The maturity of DCs was determined by detecting the number of CD80⁺ CD86⁺ DCs. As shown in Fig. S18 (Supporting information), the maturation percentage of DCs in the PBS group was 5.63%, and in the HAuHbO₂+NIR group could reach 9.65%, which was 1.71 times higher than that in the PBS group (Fig. S18a). It demonstrated that laser-mediated HAuHbO₂ promotes the maturation of DCs to present antigens to T cells for specific immune responses. Consequently, the proportion of T cells was also assayed. CD3 is often used to differentiate between helper T cells (CD3⁺ CD4⁺ Tc) and killer T cells (CD3⁺ CD8⁺ Tc). Fig. S18b shows the proportion of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ Tc in tumor tissues. The highest percentages were found in the group treated with HAuHbO₂+NIR, which was 11.42- and 6.46-fold higher than in the PBS group, respectively. The expression levels of CD4 and CD8 in tumor tissues were detected by immunofluorescence, and differences across different treatment groups were consistent with the above analysis (Fig. S19 in Supporting information). In addition to activating T cells to attack tumors, mature DCs also induced the release of interferon-γ (IFN-γ), thereby stimulating T cells to differentiate into tumor-specific IFN-γ T cells (IFN-γ⁺Tc). In Fig. S18c, the proportion of IFN-γ⁺Tc in tumor tissues in the PBS group was 3.49%. HAuHbO₂+NIR increased the proportion of IFN-γ⁺ Tc to 11.5% (3.29-fold).

In addition, a major challenge in tumor immunotherapy is the development of immune tolerance or immunosuppression by the tumors due to the fact that tumor cells release cytokines to summon Treg cells [27,28]. Therefore, the presence of Treg cells is an important indicator of tumor immunoassay [29]. The number of CD3⁺ CD4⁺ Foxp3⁺ Tc was examined. As shown in Fig. S18d, the percentage of Treg cells in the PBS group was 9.30%, and in the HAuHbO₂+NIR group decreased to 2.15%. The expression of Foxp3 in the tumor tissues of each group is also clearly shown in Fig. S19 (Supporting information), in which there was no significant expression of Foxp3 in the nanozyme group. These results indicate that nanozymes assisted by NIR laser could effectively reduce the number of Treg cells in tumor tissues. When the immune response of the organism is activated, the secretion of immune-related factors (interleukin (IL)-12, tumor necrosis factor (TNF)-α, etc.) in tumor tissues increases, activating T cells to kill tumor cells [30-32]. As shown in Fig. S20 (Supporting information), the highest levels of TNF-α, IL-12, and IFN-γ were found in the HAuHbO₂+NIR group. In conclusion, HAuHbO₂ with NIR laser irradiation can induce ICD and significantly promote the maturation of DCs to activate immunity. At the same time, it can promote the infiltration of T cells within the tumor and reduce the number of Treg cells, resulting in decreased immunosuppression.

This study reports HAuHbO₂ nanozyme that can be used as an antitumor preparation with multiple functions and catalyzed by spontaneous cascades. The specific spatial arrangement of the hollow gold rods enables the nanozyme to possess GOD-like enzymatic activity and excellent photothermal conversion ability. The loaded Hb, as a natural protein, plays a POD-like role in the nanozymatic system, providing the raw material for the Fenton reaction for CDT and promoting the antitumor therapy of the nanozyme by its own oxygen-carrying function. HAuHbO₂ displays an efficient ^•OH-generating capacity in the tumor microenvironment to induce apoptosis in tumor cells. The spontaneous cascade catalysis and the synergistic effect with PTT activate the ICD effect in tumors. By promoting DC maturation, stimulating T cell activation, inhibiting the number of Treg cells, and increasing cytokine secretion, the antitumor immune response was enhanced, ultimately inhibiting tumor growth. In addition, HAuHbO₂ can exert self-sufficiency, circulating reactants and products, and exert maximum value by making the best use of its components at the tumor site. Overall, the nanozyme reported in this study will provide great potential for clinical cancer therapy.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Dou Zhang: Writing – review & editing, Writing – original draft, Visualization, Software, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. Xinyi Cai: Methodology, Formal analysis, Data curation, Conceptualization. Yimin Li: Funding acquisition. Yong Liu: Methodology, Formal analysis. Long Qiu: Visualization, Methodology. Zhenying Diao: Formal analysis, Data curation. Xuyi Liu: Software, Methodology. Yuta Nishina: Methodology, Formal analysis, Data curation. Yajuan Zou: Methodology, Formal analysis, Data curation. Jianbo Sun: Validation, Data curation. Shujing Liang: Supervision, Funding acquisition. Daxiang Cui: Supervision, Methodology, Funding acquisition, Data curation, Conceptualization. Ting Yin: Writing – review & editing, Visualization, Supervision, Resources, Methodology, Funding acquisition, Data curation, Conceptualization.

Acknowledgments

This work was funded by Natural Science Foundation of Guangdong Province (No. 2024A1515030063), Construction Project of Nano Technology and Application Engineering Research Center of Guangdong Medical University (No. 4SG25008G), Discipline Construction Project of Guangdong Medical University (No. 4SG24015G), Funds for PhD researchers of Guangdong Medical University in 2024, National Natural Science Foundation of China (Nos. 82160478, 81703075) and Dongguan City Social Development Science and Technology Key Project (No. 20221800906362).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111569.