Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy

Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 July 2025 V1 Latest version Share on Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy Authors : Chaoran Liu , Wenyu Zhang , Qingbin He , Xinyu Ma , Jianfeng Qiu , Runxiao Zheng , and Hongjin Xue 0009-0003-9627-3299 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175246932.21694931/v1 164 views 65 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Catalyzing the glucose cascade reaction to impair tumor cell energy metabolism represents a promising strategy for tumor therapy. However, the application of natural enzymes as therapeutic agents remains limited by various challenges. Although nanozymes with multi-enzyme activities, including glucose oxidase-like (GOx-like) activity, have been reported, their development often involves complex material combinations and cumbersome synthesis processes. Here, we develop a nanozyme with GOx-, peroxidase (POD)-, superoxide dismutase (SOD)-, and NADH oxidase (NOX)-mimic activities by simply controlling the AuPt alloy ratio. The optimal cascade activity was observed for the nanozyme at an Au:Pt proportion of 13:7. Density functional theory (DFT) calculations revealed that Au sites drive glucose dehydrogenation (GOx-like), while Pt sites facilitate hydroxyl radical (•OH) generation (POD-like). Both in vitro and in vivo data indicated that Au 13 Pt 7 nanozymes disrupt tumor redox/metabolic homeostasis by depleting glucose and generating cytotoxic •OH, and impairing mitochondrial function via NOX-like, thereby inducing cell apoptosis. Notably, apoptotic immunogenic cell death (ICD) induces antitumor immunity and suppresses tumor metastasis. This study presents an innovative strategy for engineering nanozymes with multi-enzyme catalytic capabilities while demonstrates the promising application of alloy-based nanozymes in synergistic metabolic-immunotherapy. Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy Chaoran Liu 1,2# , Wenyu Zhang 1# , Qingbin He 2 , Xinyu Ma 2 , Jianfeng Qiu 2 , Runxiao Zheng 1 *, Hongjin Xue 1,2 * 1 The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan 250014, P. R. China 2 Medical Engineering and Technology Research Center, School of Radiology, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, P. R. China # These authors contributed equally to this work. * Corresponding Author. E-mail: [email protected] ; Keywords: metabolism homeostasis, alloy nanozymes, antitumor, synergistic therapy Abstract Catalyzing the glucose cascade reaction to impair tumor cell energy metabolism represents a promising strategy for tumor therapy. However, the application of natural enzymes as therapeutic agents remains limited by various challenges. Although nanozymes with multi-enzyme activities, including glucose oxidase-like (GOx-like) activity, have been reported, their development often involves complex material combinations and cumbersome synthesis processes. Here, we develop a nanozyme with GOx-, peroxidase (POD)-, superoxide dismutase (SOD)-, and NADH oxidase (NOX)-mimic activities by simply controlling the AuPt alloy ratio. The optimal cascade activity was observed for the nanozyme at an Au:Pt proportion of 13:7. Density functional theory (DFT) calculations revealed that Au sites drive glucose dehydrogenation (GOx-like), while Pt sites facilitate hydroxyl radical (•OH) generation (POD-like). Both in vitro and in vivo data indicated that Au 13 Pt 7 nanozymes disrupt tumor redox/metabolic homeostasis by depleting glucose and generating cytotoxic •OH, and impairing mitochondrial function via NOX-like, thereby inducing cell apoptosis. Notably, apoptotic immunogenic cell death (ICD) induces antitumor immunity and suppresses tumor metastasis. This study presents an innovative strategy for engineering nanozymes with multi-enzyme catalytic capabilities while demonstrates the promising application of alloy-based nanozymes in synergistic metabolic-immunotherapy. 1. Introduction Metabolic reprogramming in tumors is an important biological feature that distinguishes them from normal tissues, with abnormally active glucose metabolism being the most prominent. [1-3] They predominantly rely on aerobic glycolysis to metabolize glucose, which allows these cells to supply energy and produce the necessary building blocks to regulate tumor progression, maintenance, and metastasis, known as the “Warburg effect”. [4, 5] This distinctive metabolic trait has paved the way for the development of novel therapeutic strategies targeting key steps in glucose metabolism for antitumor treatment. [6-9] Glucose oxidase (GOx), as the core component of a biocatalytic system, can specifically catalyze glucose oxidation, generating gluconic acid and hydrogen peroxide ( H 2 O 2 ). [10, 11] This mechanism offers unique advantages in antitumor therapy: on one hand, it induces metabolic starvation by depleting glucose in the tumor microenvironment (TME); on the other, it triggers oxidative stress through H 2 O 2 accumulation. The synergistic interplay of these effects can enhance the cytotoxicity against tumor cells. However, natural enzymes are confronted with several challenges, including inadequate stability, significant costs, and a potential immunogenicity concern. [12] To overcome these bottlenecks, researchers have recently devoted efforts to developing artificial enzyme mimics with GOx-like catalytic activities, thereby providing innovative approaches for advancing next-generation tumor starvation therapy. [1, 7, 8] As a groundbreaking class of nanomaterials that can mimic the function of enzymes, nanozymes possess unique nanoscale architectures that not only endow them with outstanding catalytic activity but also confer greater stability and superior scalability compared to natural enzymes. [9, 10, 13] However, limited by complex tumor microenvironments such as hypoxia, mono GOx-like nanozymes alone struggles to completely suppress tumor growth. [14] Recently, multi-enzymatic nanozymes have demonstrated the ability to drive cascade glucose reactions and synergistically enhance antitumor effects. Luo et al. demonstrated a cascade system in which Mn-carbon dots served as the core component and ZIF-8 functioned as the shell framework, with ultrasmall Au NPs anchored onto the exterior, enabling the cascade reaction of GOx and catalase (CAT). [15] Wang et al. put forward a multifunctional nanozyme (mPt@Au) by depositing ultrasmall Au nanoparticles onto mesoporous Pt (mPt), promoting catalytic treatment through sequential biocatalytic mimetic cascades involving GOx and POD. [16] Despite these advancements, those methods often involve multistep synthesis processes, such as chemical modification, additional pre- or post-synthetic treatments, and tend to produce uneven active-component distributions, coarse interfacial structures, and insufficient electronic modulation, inevitably resulting in low activity and poor long-term durability. In recent years, noble-metal nanozymes have garnered significant attention for their exceptional catalytic activity. [17] Alloying presents a promising strategy to further expand the functional repertoire noble-metal nanozymes. [18] Of particular interest are AuPt alloys, where in the differing electron configurations and electronegativities of Au and Pt endow the electronic structures and physicochemical properties with a marked departure from those of the pure metals. [19] Herein, using a facile one-step reduction method, we developed an ultrasmall AuPt alloy nanozyme with four types of enzyme-mimicking activities — GOx, POD, SOD, and NOX. Systematic investigation revealed that the Au-to-Pt ratio in the alloy plays a critical role in modulating the enzyme-mimetic activity, among which the Au 13 Pt 7 alloy nanozymes demonstrated the highest cascade activity. To gain mechanistic insights into these catalytic processes, we conducted density functional theory (DFT) calculations that elucidate the atomic-level interactions and reaction pathways. Upon accumulation in the tumor, the Au 13 Pt 7 nanozymes possessing GOx- and POD-like activities could efficiently consume glucose to generate H 2 O 2 and then decompose it into hydroxyl radicals (•OH). The SOD-like activity could alleviate the hypoxic TME and compensate for the consumption of O 2 during the GOx -like catalysis, thus facilitating the cyclic consumption of glucose and the self-supply of H 2 O 2 substrates for •OH generation. Moreover, the inherent NOX activity can drive the oxidation of NADH to produce H 2 O 2 , which elicits a cascade of effects, including elevated reactive oxygen species (ROS) levels, disrupted mitochondrial electron transport, and diminished mitochondrial membrane potential. Thus, the Au 13 Pt 7 nanozymes can not only disturb tumor redox and metabolic balance via enzyme-mimetic cascading reactions, but also activate immunogenic cell death (ICD)-associated immune responses for cancer immunotherapy (Scheme 1). 2. Results and Discussion 2.1. Synthesis and characterization of ultrasmall AuPt nanozymes A series of ultrasmall Au x Pt 20-x alloys were prepared by a simple colloidal method with NaBH 4 as the reductant (Figure 1A). [20] The alloy ratio was regulated by adjusting the proportions of metal precursors, namely Au, Au 15 Pt 5 , Au 13 Pt 7 , Au 10 Pt 10 , and Pt. The transmission electron microscopy (TEM) images showed the spherical morphology and uniform distribution of the synthesized five nanozymes, with an average size of approximately 3 nm. (Figures 1B and S1). Moreover, the spherical aberration corrected-scanning transmission electron microscopy (AC-STEM) images of the Au 13 Pt 7 revealed a typical 5-fold twinned structure with the exposed facets predominantly composed of (111) planes of the face-centered cubic (fcc) lattice (Figure S2). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) coupled with in situ energy-dispersive X-ray spectroscopy (EDS) elemental mapping validated that Pt and Au were homogeneously dispersed throughout the nanoparticles (Figures 1C and D). The Pt content in the five nanozymes was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The gradual elevation of Pt content validated the successful fabrication of nanozymes with diverse alloy compositions (Figure 1E). The X-ray diffraction (XRD) patterns of the Au and Pt nanozymes correspond to the characteristic face-centered cubic structures of Au and Pt. For the alloy nanozymes Au 15 Pt 5 , Au 13 Pt 7 , and Au 10 Pt 10 , the diffraction peaks exhibited a systematic shift from Au (PDF#89-3697) towards Pt (PDF#70-2057), confirming the absence of phase separation and the successful formation of alloy structures (Figure 1F). X-ray photoelectron spectroscopy (XPS) was used to investigate the surface electronic states of the Au x Pt 20-x . A comparison of the Au 4f and Pt 4f peak positions in the alloy samples with those of the pure counterparts (Au or Pt) revealed a shift of approximately 1-2 eV (Figures 1G and S3). Specifically, the Au 4f peak exhibited a positive shift in binding energy, whereas the Pt 4f peak showed a negative shift. Such shifts imply electron transfer from Au to Pt, reflecting the modulation of electronic structure within the bimetallic system. [21] Above results demonstrated the successful synthesis of nanozymes with ultrasmall sizes and varying alloy ratios. Comprehensive evaluation of enzyme-mimicking activities Au and Pt nanoparticles are well-known as typical GOx-like and POD-like nanozymes, respectively. [7, 22-24] Previous studies have demonstrated that alloying can precisely modulate the catalytic activity of metals. [25-27] Therefore, we studied the enzyme-like activities of Au x Pt 20-x nanozymes and explored the correlation between the catalytic performance and alloy ratio. [25] Notably, tailoring the alloy composition allows for the fabrication of AuPt nanozymes that concurrently exhibit both GOx- and POD- activities. The GOx-like activity was determined by spectrophotometrically by monitoring H 2 O 2 production , while POD-like performance was verified via the TMB colorimetric reaction, monitoring the characteristic 652 nm absorption peak of oxidized TMB. As evidenced by Figures 2A-B and S4-5, monometallic counterparts exhibited singular enzymatic specificity (Au: GOx-like; Pt: POD-like), whereas alloyed systems demonstrated dual functionality. Among the three alloy nanozymes, Au 13 Pt 7 displayed an optimal balance of GOx-like and POD-like activities. The construction of a GOx-POD cascade reaction platform enables dual reprogramming of glucose and redox metabolism, thus enhancing therapeutic efficacy against tumors. [8, 28-30] To investigate the potential cascade reaction involving the GOx-like and POD-like activities of five nanozymes, the experiment used glucose as the initial reactant while intentionally excluded the inclusion of the POD substrate (i.e. H 2 O 2 ) within the reaction system (Figure 2C). To exclude the potential interference from oxidase-mimetic activity, which may catalyze the oxidation of TMB by O 2 (Figure S6), the TMB reaction is conducted under a N 2 atmosphere. These results indicated that Au 13 Pt 7 alloy nanozymes exhibited the highest GOx-POD-like cascade activity (Figures 2D and S7), indicating a strong correlation between activity and alloy ratio. We further validated the GOx-like and POD-like activities under varying pH conditions, which revealed that the GOx-like activity was positively correlated with pH, while the POD-like activity exhibited an inverse relationship (Figures S8-S9). This inverse pH susceptibility suggests potential for microenvironment-responsive therapeutic applications. Furthermore, the steady-state kinetic parameters of the GOx-like and POD-like activities were evaluated, including the Michaeli-Menten constant ( K m ) and maximum initial velocity ( V max ) (Figures 2E-F, S10-13). Electron spin resonance (ESR) using 5,5-dimethylpyrroline N -oxide (DMPO) as the spin trapping agent was utilized to detect •OH generation by Au 13 Pt 7 nanozymes. As shown in Figure 2G, the distinct 1:2:2:1 quartet signal characteristic of DMPO-•OH adducts confirms the efficient catalytic conversion of H 2 O 2 to •OH by Au 13 Pt 7 , underscoring their therapeutic potential for chemodynamic applications. Additional validation of this radical generation mechanism was provided by methylene blue (MB) degradation assays, where the progressive reduction of the characteristic absorption peak at 660 nm signified concurrent •OH formation (Figure S14). Given that the hypoxic microenvironment in solid tumors is inadequate to sustain the GOx-POD-mimetic cascade catalysis, we postulated that Au 13 Pt 7 nanozymes could act as SOD mimetics by catalyzing the conversion of •O 2 ⁻ to O 2 and H 2 O 2 , thus functioning as an oxygen generator. The SOD-mimetic activity was evaluated using a classical WST - 8 (i.e. Water-soluble Tetrazolium - 8) colorimetric method. WST-8 reacts with •O 2 ⁻ generated by the X/XO system to produce a formazan dye absorbing strongly at 450 nm. [31] Since this reaction step can be inhibited by SOD, the SOD-like activity can be calculated via colorimetric analysis. As depicted in Figure 2H, the •O 2 - scavenging capacity of Au 13 Pt 7 nanozymes displayed increasingly pronounced with higher concentrations, indicating the excellent SOD-like activity. After that, we also investigated the effect of Au 13 Pt 7 nanozymes on the oxidation of NADH, which not only provides the proton motive force necessary for the mitochondrial electron transport chain (ETC) but also essential for ATP production in tumor glycolysis and oxidative phosphorylation (OXPHOS) processes. [32-34] The oxidation of NADH was tracked via UV-visible absorption spectroscopy. As depicted in Figure 2I, the introduction of Au 13 Pt 7 nanozymes resulted in a pronounced decline in the characteristic absorption peak of NADH at 340 nm, along with a concomitant increase in the absorption peak of NAD⁺ at 260 nm. These spectral changes correlated closely with the conversion of NADH to NAD⁺, indicating that the Au 13 Pt 7 nanozymes could induce NADH oxidation by mimicking NOX. Crucially, during the Au 13 Pt 7 -mediated oxidation of NADH, a substantial generation of H 2 O 2 was observed, as verified by horseradish peroxidase-assisted oxidation of TMB into blue-colored TMB ox . Figure S15 shows a TMB ox peak at 652 nm with Au 13 Pt 7 , suggesting H 2 O 2 production from NADH oxidation. The generation of H 2 O 2 could further mitigate the consumption of reaction substrates in POD-mimetic catalytic processes, thus promoting the production of more •OH through an H 2 O 2 -recycling mechanism. Collectively, Au 13 Pt 7 nanozymes with GOx - , POD - , SOD - , and NOX - mimetic activities can function as a self-amplifying nanoreactor to efficiently deplete glucose and perturb redox homeostasis via cascading catalytic reactions, thereby showcasing substantial potential in reprogramming tumor energy and redox metabolism. Theoretical studies on the enzyme-like catalytic processes To elucidate why the AuPt alloys, rather than individual Au or Pt, exhibited the GOx-POD-like cascade catalytic properties, we performed DFT calculations to identify the corresponding active sites. We first compared the GOx-like kinetics of Au and Pt active sites within the Au 13 Pt 7 alloy during the catalytic glucose dehydrogenation-oxidation process. Typically, the glucose oxidation process involves an initial dehydrogenation of glucose, followed by O 2 receiving protons and electrons to form H 2 O 2 . Figures 3A-3B illustrates the geometric configurations and energy changes during dehydrogenation process of glucose catalyzed by Au 13 Pt 7 . Initially, a glucose molecule adsorbs onto the alloy surface, followed by two consecutive dehydrogenation steps that form gluconic acid while releasing 2*H. The results revealed that for the Pt sites, the rate-determining step (RDS) of the entire reaction pathway is the removal of the first H atom from glucose, exhibiting an energy barrier of 0.68 eV. In contrast, for the Au sites, both dehydrogenation-oxidation steps of glucose occur as consecutive exothermic processes. This strongly suggests that Au sites are more likely to serve as the GOx-like catalytic centers. The dynamic effects on the POD-like performance were also studied. Figures 3C-D showed the POD-like catalytic kinetics on Au and Pt active sites within Au 13 Pt 7 . Initially, an H 2 O 2 molecule adsorbs at the surface of Au 13 Pt 7 . Thereafter, the adsorbed H 2 O 2 is homogeneously dissociated to yield two •OH radicals, with subsequent desorption of a free •OH, representing the RDS of the overall reaction pathway. In this step, Pt sites significantly favor •OH generation, as evidenced by the lower energy barrier (1.37 eV) compared to Au sites (2.09 eV). The evidence strongly indicates that Pt sites are predisposed to function as the POD-like catalytic centers. In summary, GOx-POD-mimetic cascade catalytic properties of Au 13 Pt 7 can be ascribed to the coexisting active sites of both Au and Pt. In vitro antitumor effect of Au 13 Pt 7 nanozymes Given the excellent multiple enzyme-like catalytic properties of Au 13 Pt 7 nanozymes, we proceeded to investigate in vitro antitumor ability. Endocytosis and lysosomal metabolism of Au 13 Pt 7 nanozymes are essential for therapy, as endocytosis is nanoparticles’ primary cellular uptake route. [35] To investigate the mechanism of cell phagocytosis, confocal laser scanning microscopy (CLSM) was employed to assess the subcellular co-localization of the Au 13 Pt 7 nanozymes and lysosome (marked by Lyso Tracker Green). Figure 4A presents the fluorescent co-localization analysis. In the overlay image, the yellow region represents the co-localization of the Au 13 Pt 7 nanozymes and lysosome. The Pearson coefficient (PC) value quantified the degree of co-localization between the Au 13 Pt 7 nanozymes and Lyso Tracker Green. After 3 h, the value rose from 0.45 to 0.91, suggesting Au 13 Pt 7 nanozymes internalization in 4T1 cells via the endolysosomal pathway. After 6 h, red fluorescence separated from green (PC=0.58), confirming lysosomal escape of Au 13 Pt 7 nanozymes via the “ proton sponge effect ” . [36] Additionally, flow cytometry analysis of phagocytosis demonstrated that the phagocytosis process was time-dependent and exhibited a co-localization trend consistent with that observed in CLSM (Figure S16). To assess the in vitro therapeutic efficacy of Au 13 Pt 7 nanozymes (endowed with multiple enzymatic activities), 4T1 cells were cultured with different concentrations of Au 13 Pt 7 nanozymes, along with Au and Pt control groups. Cell viability was subsequently evaluated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) assay. As shown in Figure 4B, Au, Pt and Au 13 Pt 7 nanozymes showed different degrees of concentration dependent cytotoxicity, and Au 13 Pt 7 nanozymes showed the strongest effect on 4T1 cells. Subsequently, to substantiate the favorable biological safety of the nanozymes, mouse embryonic fibroblasts (NIH3T3) were utilized to assess the cytotoxicity of the Au 13 Pt 7 nanozymes. As shown in Figure S17, even at a concentration of 200 μ g mL -1 , the viability of NIH3T3 cells remained as high as 90%, indicating relatively low cytotoxicity of Au 13 Pt 7 nanozymes toward normal cells. Following the treatment of cells with a mixture of Calcein AM and PI, viable cells emitted intense green fluorescence, whereas non-viable cells exhibited intense red fluorescence. Au 13 Pt 7 nanozymes showed peak red fluorescence in CLSM (Figure 4C), revealing maximal apoptosis due to enhanced multi-enzyme cascade cytotoxicity. The same trend in apoptosis was further observed via quantitative flow cytometry analysis (Figure 4D). The apoptosis rate in the Au 13 Pt 7 nanozymes group was 56.8%, which was markedly higher than that in the PBS group (3.2%), Au group (26.4%), and Pt group (39.1%). The above results indicated that Au 13 Pt 7 nanozymes enhanced the anti-tumor effect. We hypothesize that Au 13 Pt 7 nanozymes antitumor activity arises from their multi-enzymatic properties: disrupting tumor energy metabolism via glucose depletion and ATP inhibition, and destabilizing redox homeostasis through enzymatic cascades. Thereafter, since mitochondrial dysfunction is a key hallmark of apoptosis, mitochondrial membrane potential (MMP) in treated 4T1 cells was assessed using a JC-1 kit. When mitochondrial function deteriorates, the integrity of the mitochondrial membrane is disrupted, resulting in a decrease in MMP . [37] Under such circumstances, JC-1 exists as a monomer, emitting green fluorescence. Notably, the Au 13 Pt 7 nanozymes group exhibited a higher level of JC-1 monomers compared to other groups, indicating MMP collapse (Figure 4E). This impaired H⁺ transport (matrix to intermembrane space) was due to suppression of NADH-dependent oxidative phosphorylation. [38] To gain a more in-depth understanding of the synergistic mechanism of enzymatic cascade reactions that induce 4T1 cell death, we measured intracellular ROS levels and heme oxygenase-1 (HO-1) expression . [39, 40] Designed as multi-enzyme mimics, Au 13 Pt 7 nanozymes concurrently exhibit GOx/SOD-like activities to deplete glucose and produce H 2 O 2 , while their POD-like activity catalyzes H 2 O 2 into cytotoxic hydroxyl radicals, collectively elevating ROS levels in tumor microenvironments. In order to verify the effectiveness, the intracellular ROS levels in 4T1 cells across different treatment groups were quantified using the fluorescent ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). As shown in Figure 4F, in 4T1 cells treated with PBS and nanozymes of different formulations (Au, Pt and Au 13 Pt 7 nanozymes), Au 13 Pt 7 nanozymes treated cells showed significantly higher ROS levels. We further evaluated ROS production in different treatment groups by flow cytometry analysis, with the Au 13 Pt 7 nanozymes group displaying the strongest fluorescence signal (Figure 4G). Building upon the outstanding ROS generation capacity of Au 13 Pt 7 , an in-depth exploration of the redox equilibrium at the cellular level was conducted. We incubated 4T1 cells with PBS, Au, Pt, or Au 13 Pt 7 nanozymes for 24 h and then adopted western blot to measure heme oxygenase-1 (HO-1) expression levels. Both Pt and Au 13 Pt 7 nanozymes significantly increased HO-1 expression, indicating they amplify the oxidative stress response in tumor cells (Figure 4H-4I). In vitro induction of ICD by combination therapy ICD is mediated by the liberation of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) from necrotic tumor cells. These tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), which are mainly composed of high-mobility group box 1 protein (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP), enable the recruitment and maturation of antigen-presenting cells (APCs). [41-43] In the late phase of apoptosis, HMGB1 is released from damaged or disrupted cell nuclei into the cytoplasm. [44] As depicted in Figure 5A, in the PBS treated group, HMGB1 exhibited predominantly nuclear localization of its green fluorescence in 4T1 cells. Notably, in the group treated with Au 13 Pt 7 nanozymes, green fluorescence was scarcely detectable, likely attributable to the complete rupture of the plasma membrane. An early event in immunogenic cell death (ICD) is the translocation of calreticulin (CRT) from the ER to the plasma membrane, where it acts as an ”eat me” signal. This translocation enables APCs to recognize and engulf the dying cells, thereby initiating an immune response. Alexa 488-CRT staining revealed intense plasma membrane fluorescence in Au 13 Pt 7 -treated 4T1 cells, demonstrating CRT translocation (Figure 5B). Flow cytometry-based quantitative analysis of CRT expression demonstrated that the expression level of CRT-positive cell population of the Au 13 Pt 7 nanozymes group was significantly higher than that in PBS groups (Figure 5C). By the apoptotic vesicle phase, cancer cells release ATP, functioning as a ”find me” signal for dendritic cell (DC) precursors. The ATP concentrations in 4T1 cells treated with PBS, Au, Pt, and Au 13 Pt 7 nanozymes were measured using a specific ATP detection assay. Results showed the lowest intracellular ATP concentration in Au 13 Pt 7 nanozymes-treated cells, indicating higher extracellular ATP release (Figure 5D). These results indicate that Au 13 Pt 7 nanozymes can induce ICD, which is beneficial for tumor immunotherapy. Furthermore, ICD can induce the release of strong immunogenic antigens, leading to the full maturation and infiltration of DCs. The maturity of bone marrow-derived dendritic cells (BMDCs) was quantified using flow cytometry, based on the percentage of CD11c⁺CD80⁺CD86⁺ cells. As shown in Figures 5E-5F, compared with other groups, the maturity rate of BMDCs in the Au 13 Pt 7 groups were as high as 44.2%. Au 13 Pt 7 nanozymes enhance DC maturation by releasing immunogenic antigens that promote antigen processing and activation. In conclusion, these results verified that the release of TAAs and DAMPs enhanced by Au 13 Pt 7 nanozymes greatly facilitates the recruitment and maturation of DCs (Figure 5G). CT imaging and in vivo antitumor effect of Au 13 Pt 7 nanozymes To improve the targeting efficiency and biocompatibility of Au 13 Pt 7 nanozymes, the tumor-targeting molecule folic acid (FA) was conjugated onto their surface. [25, 45, 46] Employing a strategy analogous to polyethylene glycol (PEG) modification, the Au 13 Pt 7 nanozymes surface was coated with thiol-terminated PEG-FA (SH-PEG-FA). Fourier transform infrared (FTIR) and UV- vis absorption spectroscopy verified the successful functionalization of FA. In the FTIR spectrum, the peak at 1607 cm -1 corresponded to the NH bending vibration of FA (Figure S18). Figure S19 shows the UV- vis absorption peak at 285 nm, corresponding to the characteristic absorption of the methotrexate ring in FA. [25] No significant change in enzyme-like activity was observed with FA modification (Figures S20-S21). Integrated diagnosis and therapy could enhance patient outcomes by enabling real-time disease monitoring, personalized treatment optimization, and reduced side effects. [47-49] Because of the potent X-ray attenuation capabilities of Au and Pt (5.16 and 4.99 cm -2 kg -1 at 100 keV, respectively), the Au 13 Pt 7 nanozymes could be used for CT（Computed Tomography） contrast agents. We conducted CT imaging using a subcutaneous 4T1 tumor-bearing mouse model. When the tumor volume reached approximately 100 mm 3 , mice with 4T1 tumors were randomly assigned to five groups (n=5 per group): (1) PBS, (2) Au, (3) Pt, (4) Au 13 Pt 7 , and (5) Au 13 Pt 7 -FA nanozymes. On days 0, 2, and 4, each group was intravenously injected with the corresponding nanozymes (or PBS) via the tail vein for imaging monitoring and data recording (Figure 6A). The CT imaging performance of Au 13 Pt 7 -FA were systematically evaluated through in vitro and in vivo experiments. In vitro CT imaging revealed concentration-responsive visibility of Au 13 Pt 7 -FA (Figure 6B). A positive correlation was observed between CT signal intensity (in Hounsfield units, HU) and the concentration of Au 13 Pt 7 -FA. To enable more intuitive in vivo imaging, we conducted imaging studies following injections of Au 13 Pt 7 -FA at different time points. [50] The in vivo imaging results revealed no significant difference in CT signal intensity between tumor sites and surrounding tissues prior to nanozyme administration. In contrast, after the injection, the tumor area became significantly brighter, indicating substantially increased X-ray absorption (Figure S22). At selected post-injection time points, tumor imaging was subsequently performed following administration of Au 13 Pt 7 -FA (1 mM, 100 μL). (Figure 6C: coronal view; Figure S23: axial view). Notably, both CT values and signal intensities exhibited significant enhancement within 5 min post-injection and remained stable for 60 min (Figure S24). Au 13 Pt 7 -FA shows potential for enabling more comprehensive tumor characterization, as demonstrated by these findings. Inspired by the promising in vitro anti-tumor activity of Au 13 Pt 7 nanozymes and their favorable CT imaging performance, in vivo therapeutic efficacy was further investigated using 4T1 tumor-bearing mice. We examined the biodistribution of Au 13 Pt 7 -FA in various organs and tumor tissues. As illustrated in Figure 6D, tumor sites in the Au 13 Pt 7 -FA group demonstrated rapid accumulation within 1 h post-intravenous injection. The Cy5 fluorescence intensity reached its peak at 36 h after administration in the Au 13 Pt 7 -FA group, followed by a time-dependent decrease, suggesting gradual systemic clearance of the Au 13 Pt 7 -FA nanozymes. In contrast, free Cy5 showed rapid renal excretion following intravenous injection. Furthermore, Figure 6E illustrates that the Au 13 Pt 7 -FA nanozymes accumulated significantly more in tumor tissues compared to the free Cy5 group. Collectively, these findings corroborate that FA-functionalized Au 13 Pt 7 nanozymes possess excellent tumor-targeting capabilities. Subsequently, a comprehensive toxicological evaluation was conducted to ensure the biosafety. As depicted in Figure 6F, the hemolysis rates of the Au 13 Pt 7 -FA nanozymes at various concentrations were consistently below 5%, suggesting their suitability for intravenous administration. Following this, serum biochemistry and blood routine analyses revealed that parameters related to blood composition, liver function, and kidney function remained within normal reference ranges (Figures 6G-6H; Figure S25). To further evaluate potential toxic effects, hematoxylin and eosin (H&E) staining was performed on multiple organs. Histological analysis revealed excellent histocompatibility and no discernible toxic effects on major organs (Figure S26). Throughout the monitoring period, no significant difference in body weight was observed between mice injected with Au 13 Pt 7 -FA nanozymes and those injected with PBS (Figure S27). Taken together, these experimental findings demonstrate that Au 13 Pt 7 -FA nanozymes exhibit excellent in vivo biosafety. Given their promising demonstrated tumor inhibition in vitro , selective tumor accumulation in vivo , and biosafety, we investigated the in vivo antitumor effects of Au 13 Pt 7 -FA nanozymes. Based on tumor growth curves and weights, both Au 13 Pt 7 and Au 13 Pt 7 -FA nanozymes significantly inhibited tumor growth relative to other groups, with Au 13 Pt 7 -FA nanozymes exhibiting a more pronounced inhibitory effect (Figure 6I). The harvested tumor tissues were weighed and imaged, showing results consistent with the tumor volume trend (Figures 6J-6K). To further characterize the anti-tumor effects and dissect the underlying mechanisms, tumor samples were collected for histological analysis. Hematoxylin and eosin (H&E) staining showed maximal tumor cell reduction, widened intercellular spaces, and nuclear pyknosis in Au 13 Pt 7 -FA groups, indicating superior tumor-killing efficacy (Figure 6L). Then, we explored the immune mechanism underlying their anti-tumor effects. Flow cytometry analysis was employed to quantify the proportions of CD4 + and CD8 + T lymphocytes within tumor tissues following treatment with PBS, Au, Pt, Au 13 Pt 7 and Au 13 Pt 7 -FA nanozymes. Levels of CD8 + T cell infiltration were 1.4%, 1.9%, 3.3%, 5.4%, and 7.7%, respectively, alongside CD4 + T cells infiltration rates of 12.2%, 18.4%, 22.4%, 26.4% and 30.1% (Figure 6M). To further investigate the systemic remodeling of immune responses in mice, the ratios of T cells in lymphoid tissues (including lymph nodes and spleens) were examined. Flow cytometry analysis of lymph nodes draining the tumor (Figure S28) demonstrated the presence of substantial CD4⁺ and CD8⁺ T cells populations in Au 13 Pt 7 -FA nanozymes-treated groups, indicative of enhanced recruitment of lymph node-associated immune effector cells. Similarly, splenic tissues showed significant upregulation of both CD4 ⁺ and CD8 ⁺ T cells (Figure S29). To investigate whether Au 13 Pt 7 -FA nanozymes treatment can induce DCs maturation in tumor, flow cytometry was employed to assess the quantity of mature DCs (Figure S30). In lymph node analysis, the Au 13 Pt 7 -FA nanozyme-treated group exhibited the most significant upregulation of CD80 and CD86 expression, indicating effective DCs maturation (Figure 6N). In summary, Au 13 Pt 7 -FA nanozymes-mediated tumor metabolic reprogramming synergistically enhanced ICD effect, which facilitated APC recruitment, maturation, and antigen uptake/presentation, thereby driving adaptive T cell-mediated anti-tumor responses. Conclusion In this study, we developed ultrasmall noble metal alloy nanozymes with quadruple enzyme-mimicking activities (GOx, POD, SOD, NOX) by simply controlling the AuPt alloy ratio, with Au 13 Pt 7 exhibiting optimal cascade catalytic performance. The Au 13 Pt 7 nanozymes reprogram tumor metabolism and redox homeostasis by depleting glucose, self-supplying H 2 O 2 , generating •OH, and disrupting mitochondrial function, leading to apoptosis and ICD. The latter promotes DCs maturation and T-cell infiltration, synergizing metabolic therapy with antitumor immunity. In vitro and vivo evaluations confirmed potent tumor cell killing and significant antitumor efficacy, along with CT imaging capability. This work establishes a self-sustaining catalytic platform for synergistic metabolic-immunotherapy against tumors with complex microenvironments. Future work should explore broader alloy compositions to fine-tune enzymatic activities and assess clinical translation potential in combination with established immunotherapies. Acknowledgments This study was supported by the National Natural Science Foundation of China (22207066), Shandong Provincial Natural Science Foundation (ZR2023QB273, ZR2024QB141), Traditional Chinese Medicine Science and Technology Project of Shandong Province (Q-2022142, Q-2023096), Shandong Pharmaceutical and Health Science and Technology Project (202420000958), Science and Technology Innovation Development Program of Taian (2023NS100) and Project for Scientific Research Innovation Team of Young Scholars in Colleges and Universities of Shandong Province (2022KJ196). Conflict of Interest Statement The authors declare no competing financial interest or per-sonal relationships that could have appeared to influence thework reported in this paper. Yuanyu Huang is a member of theExploration editorial board. 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(C) HAADF-STEM image and (D) corresponding elemental mapping of Au 13 Pt 7 nanozymes. (E) The Au content in five AuPt alloy nanozymes determined by ICP-OES (n = 3 independent experiments). (F) XRD patterns of the synthesized AuPt nanoalloys with five distinct compositions. (G) The XPS spectra of the five as-synthesized AuPt nanoalloys with tunable compositional ratios. Figure 2 In vitro evaluation of the multi-enzyme-like activities of AuPt alloy nanozymes. (A) Comparison of the relative glucose oxidase activity among five AuPt nanoalloys with different compositions. (B) Comparison of the relative peroxidase-like activity among five AuPt nanoalloys with different compositions. (C) Comparative evaluation of the GOx-POD cascading enzymatic performance in five nanoalloy systems. (D) Schematic illustration of the GOx-POD cascade activity of AuPt nanozymes. Kinetic evaluation of the GOx-like (E) and POD-like (F) activities of Au 13 Pt 7 nanozymes (using glucose and H 2 O 2 as substrates, respectively). (G) ESR spectra of ·OH in H 2 O 2 and Au 13 Pt 7 + H 2 O 2 groups. (H) Evaluation of SOD-like activity of Au 13 Pt 7 nanozymes at varying concentrations. (I) Evaluation of the NOX-like activity of the Au 13 Pt 7 nanozymes, where NADH shows a characteristic absorption peak at 340 nm. Figure 3 Computational study on the enzymatic activity of the Au 13 Pt 7 nanozymes. (A) Geometric configurations and energy changes (B) during glucose dehydrogenation catalyzed by the Au 13 Pt 7 nanozymes. (C) Geometric configurations and energy changes (D) during the peroxidase-like reaction catalyzed by the Au 13 Pt 7 nanozymes. Figure 4 In vitro evaluation of the antitumor activity of Au 13 Pt 7 nanozymes. (A) Colocalization CLSM images of Au 13 Pt 7 nanozymes and 4T1 cells after different time incubations (1, 3, and 6 h). (B) Cytotoxicity assays of 4T1 cells with various treatments of Au 13 Pt 7 nanozymes at varied concentrations (0,12.5, 25, 50, 100, and 200 μM). (C) CLSM images of AM/PI stained 4T1 cells treated with different conditions. (D) Flow cytometry analysis of the apoptosis of 4T1 cells subjected to different treatments: PBS, Au, Pt, and Au 13 Pt 7 . (E) CLSM images staining mitochondria treated with different conditions. CLSM images (F) and flow cytometry analysis (G) of intracellular ROS level under different treatment conditions. (H-I) Expression of HO-1 protein in 4T1 cells under different treatment conditions. Figure 5 Immunogenic cell death (ICD) induction in vitro . (A) Immunofluorescence staining (green: HMGB1, blue: DAPI) and corresponding fluorescence intensity in 4T1 cells after various treatments. (B) Fluorescence images of calreticulin (CRT) in 4T1 cells across different treatment groups and the corresponding flow cytometric evaluation (C). (D) ATP levels in 4T1 cells across different treatment groups. (E-F) Matured DCs (CD80 + CD86 + ) were analyzed by flow cytometry after different treatments with quantitative analysis. (G) Schematic illustration of Au 13 Pt 7 nanozymes-triggered immunogenic cell death for enhanced immunotherapy. Figure 6 Functional validation of in vitro and in vivo imaging capabilities and evaluation of in vivo therapeutic efficacy mediated by Au 13 Pt 7 nanozymes. (A) Schematic illustration for tumor treatment schedule. (B) In vitro CT images of Au 13 Pt 7 nanozymes with varying concentration gradients and their correlation with Hounsfield unit (HU) values. (C) In vivo CT images of 4T1-tumor-bearing nude mice at 0, 5, 10, 30, and 60 min after intratumor injecting Au 13 Pt 7 nanozymes (1 mM, 100 μL). (D) In vivo fluorescence imaging and ex vivo organ (E) imaging of mice treated with Au 13 Pt 7 -FA-Cy5 and free Cy5 at different time points. (F) Hemolysis analysis and corresponding digital photograph of blood incubated with water and different concentrations of Au 13 Pt 7 nanozymes. (G-H) Biochemical blood analysis of BALB/c mice across treatment groups revealed hepatic and renal function profiles, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA). (I-J) Tumor weight and digital photographs (K) of representative excised tumors in each group after treated with 14 days. (L) H&E staining of tumor slices in different groups after 14 days. (M) Flow cytometric quantification of CD4 + and CD8 + T lymphocyte subsets in tumor-draining lymph nodes (TDLNs) was performed to characterize tumor-specific immune cell infiltration profiles. (N) Flow cytometric analysis was conducted to evaluate the maturation status of dendritic cells (DCs) in tumor tissues through quantitative detection of surface maturation markers (CD80/CD86). The table of contents: Through precise control of the alloy ratio, this study introduces an ultrasmall AuPt alloy nanozyme exhibiting quadruple enzyme-like activities. The multifunctional nanozymes initiate a distinctive catalytic cascade that disrupts tumor metabolic and redox homeostasis, inducing robust immunogenic cell death (ICD). The ICD and tumor antigen release trigger potent antitumor immune responses, demonstrating synergistic metabolic-immunotherapy efficacy. Keywords: metabolism homeostasis, alloy nanozyme, antitumor, synergistic therapy Chaoran Liu, Wenyu Zhang, Qingbin He, Xinyu Ma, Jianfeng Qiu, Runxiao Zheng *, Hongjin Xue* Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy Supplementary Material File (image8.emf) Download 1.42 MB Information & Authors Information Version history V1 Version 1 14 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords alloy nanozymes metabolism homeostasis synergistic therapy Authors Affiliations Chaoran Liu Shandong Provincial Qianfoshan Hospital View all articles by this author Wenyu Zhang Shandong Provincial Qianfoshan Hospital View all articles by this author Qingbin He Shandong First Medical University View all articles by this author Xinyu Ma Shandong First Medical University View all articles by this author Jianfeng Qiu Shandong First Medical University View all articles by this author Runxiao Zheng Shandong Provincial Qianfoshan Hospital View all articles by this author Hongjin Xue 0009-0003-9627-3299 [email protected] Shandong Provincial Qianfoshan Hospital View all articles by this author Metrics & Citations Metrics Article Usage 164 views 65 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Chaoran Liu, Wenyu Zhang, Qingbin He, et al. Ultrasmall AuPt Nanozymes with Multienzyme-Mimetic Cascade Catalysis for Synergistic Tumor Metabolic-Immunotherapy. 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