Synergistic disruption of redox and energy homeostasis via high-entropy layered double hydroxide nanozymes for enhanced tumor therapy

Summary

The efficacy of nanozyme catalytic therapy is often affected by the complexity of tumor microenvironment (TME), since tumors use energy metabolism to resist oxidative stress. Relying only on disrupting the redox balance is usually not enough to completely remove the tumor. To address this problem, a high-entropy engineering strategy was developed to build a Pt-LDH nanozyme, where Pt clusters are anchored onto a high-entropy layered double hydroxide (LDH) support. This design endows the Pt-LDH with glucose oxidase (GOx)-like activity, which directly consumes intratumoral glucose to cut off the energy supply, overcoming the limitation of insufficient endogenous oxidants and stimulated subsequent oxidative stress. By simultaneously disrupting the energy metabolism and homeostasis, the Pt-LDH nanozyme achieves a potent synergistic therapeutic effect. Both in vitro and in vivo studies showed that Pt-LDH inhibited tumor growth with minimal toxicity. This proves that high-entropy design can be a powerful and practical way to build nanozyme for cancer therapy.

Graphical abstract

Highlights

• •We constructed Pt-LDH, which can dual disruption of the redox and the energy steady state
• •Pt-LDH demonstrates a higher V_max than the majority of reported LDH-based nanozymes

molecular biology; materials science

Introduction

The tumor microenvironment (TME) is illustrated by hypoxia, immunosuppression, and a distinct metabolic profile, all of which conspire to thwart effective cancer therapy.^1,2,3 While reactive oxygen species (ROS)-related therapies have shown promise, they are severely impeded by two fundamental limitations.⁴ Initially, the hypoxia within the TME limits the generation of oxygen dependent radicals.⁵ While various catalase (CAT)-mimicking nanozymes have been developed to relieve hypoxia by decomposing endogenous H₂O₂, the limited intracellular H₂O₂ pool often leads to “fuel exhaustion” crisis, rendering the therapy unsustainable.^6,7 In addition, and perhaps more importantly, tumors possess a metabolic plasticity.⁸ Even in oxidative stress, cancer cells can rapidly upregulate their energy metabolism to fuel repair and antioxidant defense systems, and neutralize the therapeutic damage.^9,10 Thus, it is necessary to shift from simple ROS generation to a comprehensive strategy that simultaneously sever the tumor’s energy supply and amplifies oxidative stress.

High-entropy nanomaterials have recently emerged as promising nanozyme candidates due to their unique multi-element composition, which creates a highly disordered lattice structure and tailored electronic environment, favorable for multi-enzyme cascade activities.^11,12,13 By combing five or more elements into one single lattice, high-entropy engineering induces severe lattice distortion and unique electronic interactions with high active sites that are not available in monometallic systems.^14,15 We hypothesized that high-entropy layered double hydroxides (denoted as HE-LDHs), with their tunable transition metals and large specific surface area could be used for multi-enzyme activities.^16,17 If we could engineer HE-LDH to have both glucose oxidase (GOx)-like and peroxidase (POD)-like activities, then a self-reinforcing therapeutic cascade: glucose to stop the supply of energy (starvation) and fresh H₂O₂ to fuel ROS oxidation.

Herein, we prove this hypothesis by constructing a Pt-decorated high-entropy LDH nanozyme (denoted as Pt-LDH) using a simple high-entropy engineering strategy (Scheme 1). Unlike traditional designs with microstructure uncontrollable or single-functionality, we exploit the high-entropy support and Pt clusters, which maximizes the atomic utilization efficiency, and optimizes the electronic structure to drive a powerful starvation-oxidation synthesis. By utilizing intratumoral glucose and unleashing ROS storm, Pt-LDH overcomes the metabolic adaptability of cancer cells, and achieves superior antitumor activity with negligible systemic toxicity. This work establishes high-entropy design as an avenue for the next generation intelligent nanomedicines.

Scheme 1.

Schematic of Pt–LDH induced cancer therapy

Results and discussion

Design and characterizations

The fabrication process of Pt-LDH is illustrated in Figure 1A. In brief, high-entropy LDH were fabricated using a co-precipitation method, the metal precursors were mixed uniformly, followed by the deposited Pt nanocluster on the HE-LDH matrix.

Figure 1.

Design and characterizations of Pt-LDH

(A) Schematic illustration of the construction of Pt-LDH.

(B) TEM images of Pt-LDH.

(C) AFM image of Pt-LDH.

(D) The XRD pattern of Pt-LDH, Ir-lDH, and HE-LDH.

(E) Elemental mappings of Pt, Fe, Ni, Cu, Mn, and Co elements in Pt-LDH.

(F) XPS survey spectra of Pt-LDH.

(G) Pt 4f XPS spectra of Pt-LDH.

(H) Ir 4f XPS spectra of Ir-LDH. Scale bars, (B) 100 nm (E) 900 nm.

While the iridium-decorated HE-LDH was regarded for the comparison. Initially, Transmission electron microscopy (TEM) images (Figures 1B and S1) reveal that both Pt-LDH and Ir-LDH exhibit two-dimensional nanosheet morphologies (∼80 ± 5 nm in lateral size) uniformly decorated with nanoclusters. As shown in Figure S2, the high Zeta potential of +33.1 mV, +21.2 mV, and +31.2 mV observed for HE-LDH, Pt-LDH, and Ir-LDH suggesting good dispersion stability. The thickness of the samples was subsequently investigated via atomic force microscopy (AFM). AFM measurements indicate that Pt-LDH exhibits a thinner nanosheet morphology (∼5.81 nm) (Figure 1C) compared to Ir-LDH (∼8.58 nm) (Figure S3). Subsequently, the crystal structures of HE-LDH, Pt-LDH and Ir-LDH were examined by X-ray diffraction (XRD), respectively. As shown in Figure 1D, the characteristic peaks of HE-LDH appear at 11.65°, 23.42°, and 33.22°, respectively, confirming the successful formation of the typical layered LDH structure.¹⁸ Notably, no obvious other peaks were detected in both Pt-LDH and Ir-LDH, likely due to the ultralow cluster loading (<5 wt %), which falls below the detection limit of XRD.¹⁹ Additionally, energy-dispersive X-ray spectroscopy (EDS) elemental mapping (Figure 1E) demonstrates the homogeneous distribution of Pt, Fe, Co, Ni, Cu, and Mn across the nanosheets. Similarly, EDS analysis of HE-LDH and Ir-LDH (Figures S4 and S5) confirms the uniform dispersion of other elements. The results provide clear evidence for the proposed synthesis strategy to successfully synthesize Pt clusters loaded quinary high-entropy nanosheet. Inductively coupled plasma optical emission spectrometry (ICP-OES) was employed to quantify the elemental composition of both materials. As summarized in Table S1, the molar ratios of Cu:Mn:Co:Ni:Fe in Pt-LDH and Ir-LDH are in good agreement with the designed high-entropy configuration. In addition, the stability of Pt-LDH and Ir-LDH were investigated via UV-spectrum. as revealed in Figure S6, no obvious fluctuation can be found after the incubation in the phosphate-buffered saline (PBS) solution for 7 days, indicating good stability.

Subsequently, X-ray photoelectron spectroscopy (XPS) survey spectra (Figures 1F and S7) was utilized to further verify the coexistence of Pt, Ir, Fe, Co, Ni, Cu, and Mn in Pt-LDH and Ir-LDH, respectively. High-resolution XPS analysis was conducted to probe the electronic states of Pt and Ir. As displayed in Figure 1G, the peaks at 71.1 eV and 74.5 eV are assigned to Pt⁰, while those at 72.4 eV and 75.8 eV correspond to Pt²⁺, indicating the coexistence of metallic Pt⁰ and oxidized Pt²⁺ species. Similarly, the Ir 4f spectrum of Ir-LDH exhibits two valence states, with peaks at 60.4 eV and 63.7 eV corresponding to Ir⁰, while signals at 61.9 eV and 65.2 eV are assigned to Ir⁴⁺, indicating a mixed Ir⁰/Ir⁴⁺ valence state (Figure 1H). The coexistence of mixed valence states is critically important for the multi-enzyme catalytic performance of Pt-LDH, which suggests a greater density of electron-rich active sites available for direct substrate activation. Additionally, the high-entropy LDH support plays a synergistic role by inducing charge redistribution at the metal-support interface, which helps stabilize the mixed valence states and further optimize the electronic structure of the anchored Pt clusters. These findings imply that both Pt and Ir are presented as clusters on the LDH surface.

Integrated investigation of enzyme-catalyzed properties

Inspired by the multi-valence of the samples, the enzyme-catalyzed activity was further investigated (Figure 2A). Specifically, the O₂ generation of various samples was investigated. Initially, the Pt-LDH group exhibits a much faster and greater increase in O₂ levels over time compared to the Ir-LDH and PBS groups. This result suggests that Pt-LDH has a superior ability to produce O₂. As expected, the O₂ production was found to be dependent on H₂O₂ concentration (Figures 2B and S8). Afterward, Figure 2C presents a quantitative analysis of the specific enzymatic activities of CeO₂, HE-LDH, Ir-LDH, and Pt-LDH, respectively. The Pt-LDH group shows a significantly higher specific activity than the other groups, being approximately 5.02 and 2.31 times that of HE-LDH and Ir-LDH, respectively. This reveals that Pt-related materials have a stronger ability to decompose H₂O₂, which is beneficial for regulating the intracellular H₂O₂ concentration and further easing the hypoxia condition in the TME. Meanwhile, the POD-like activity was analyzed to verify if this Pt-LDH group can generate ⋅OH under the TME. Figures 2D and S9 demonstrated the time-dependent absorbance changes of oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB) among HE-LDH, Ir-LDH and Pt-LDH samples. Pt-LDH exhibits a more rapid absorbance increase compared to HE-LDH and Ir-LDH, indicating the high POD-like catalytic activity of Pt-LDH. Moreover, the POD-like activity was further verified by electron spinning resonance (ESR), using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a trapping agent. DMPO selectively binds ⋅OH to form DMPO-⋅OH, as evidenced by the typical 1:2:2:1 signal line in the ESR spectra (Figure 2E). To quantitatively scrutinize specific POD-like enzyme activity of three samples, the Michaelis-Menten kinetics analysis was conducted afterward. The resulting kinetic parameters (V_max = 5.17 × 10⁻⁷ M s⁻¹, K_m = 0.4 mM) of Pt-LDH reveal the high catalytic efficiency and moderate substrate affinity compared with HE-LDH (V_max = 2.18 × 10⁻⁷ M s⁻¹, K_m = 0.42 mM) and Ir-LDH (V_max = 3.21 × 10⁻⁷ M s⁻¹, K_m = 0.78 mM) (Figures 2F and S10)., Pt-LDH demonstrates comparable enzyme activity compared with other reported nanozymes (Table S2).

Figure 2.

Integrated investigation of enzyme-catalyzed properties of Pt-LDH

(A) Schematic illustration of homeostasis disruption.

(B) O₂ production from the H₂O₂ decomposition catalyzed by Pt-LDH with different concentrations.

(C) CAT-like enzyme activity of different nanozymes (Mean ± SD, n = 3).

(D) Time-dependent oxidation of TMB by Pt-LDH.

(E) ESR spectra of DMPO/⋅OH treated with different nanozymes.

(F) Steady-state kinetics of Pt-LDH with different H₂O₂ concentrations (Mean ± SD, n = 3).

(G) Time-dependent oxidation of DPBF due to ¹O₂ generation by different nanozymes.

(H) ESR spectra of TEMP/¹O₂ treated with different samples.

(I) GOx-like enzyme activity of different nanozymes (Mean ± SD, n = 3).

(J) Time-dependent absorption of NADH during 30 min of the catalysis reaction by Pt-LDH. Statistical significance was evaluated using student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

The generation of ¹O₂ can be further confirmed by ultraviolet-visible (UV-Vis) spectra with 1,3-diphenylisobenzofuran (DPBF) as indicator, which can be degraded by ¹O₂. As shown in the time-dependent absorbance curves (Figures 2G and S11), Pt-LDH induces a more rapid decrease in absorbance than Ir-LDH and HE-LDH, indicating the superior catalytic activity of Pt-LDH. To further prove if this Pt-LDH can catalyze the production of ⋅O₂⁻ from O₂ with oxidase (OXD)-like activity, the ESR was further examined. As displayed in Figure S12, the typical peaks of ⋅O₂⁻ can be observed after treatment with Pt-LDH, Ir-LDH, and HE-LDH. The signal strength of Pt-LDH is significantly higher than that of Ir-LDH and HE-LDH, indicating its excellent OXD-like enzymatic performance, and subsequently, further detected the generation of ¹O₂, as revealed in Figure 2H, the typical 1:1:1 peak can be attributed to the ¹O₂.

To evaluate the effect of Pt-LDH on tumor cell metabolism, its GOx-like activity was examined. Pt-LDH, Ir-LDH and HE-LDH exhibit remarkable GOx-like activity, with Pt-LDH showing an outstanding catalytic performance (Figure 2I). In summary, Pt-LDH can catalyze substrates such as glucose and H₂O₂ to further generate ROS. Meanwhile, it participates in the oxidation of NADH to NAD⁺ (Figures 2J and S12). As NADH is the primary electron donor in the mitochondrial electron transport chain (ETC), its catalytic depletion directly impairs electron flux. This disruption triggers severe mitochondrial dysfunction through two interconnected pathways: first, electron leakage from the impaired ETC paradoxically generates abundant superoxide radicals (⋅O₂⁻), further exacerbating the intracellular ROS storm; second, it leads to the collapse of the mitochondrial membrane potential (ΔΨm) and subsequent ATP depletion. Importantly, this NADH oxidation works in concert with the GOx-like activity to create a dual metabolic blockade—cutting off both upstream glucose supply and downstream electron carriers. This disruption of the intracellular redox balance is crucial for interfering with cellular homeostasis, providing a theoretical basis for the subsequent catalytic activity studies.

Inspired by the excellent multi-enzyme-mimicking activities of Pt-LDH and its metabolism-inhibiting effect, the potential in vitro toxicity was further evaluated; the scheme was illustrated in Figure 3A. Firstly, a standard cell counting kit-8 (CCK-8) assay was used to investigate the in vitro antitumor effect of Pt-LDH, Ir-LDH and HE-LDH under various concentrations. As shown in Figure 3B, with the increase concentration of Pt-LDH, the viability was obvious decreased, compared to the other samples, attributing the good multi-enzyme-mimicking activities of Pt-LDH and its metabolism-inhibiting effect. To further estimate the intracellular ROS generation capacity of the three samples, confocal laser scanning microscopy (CLSM) images were characterized.²⁰ The results show that when treated with HE-LDH and Ir-LDH alone, only a weak fluorescence intensity can be detected. It is noteworthy that the Pt-LDH group exhibited higher green fluorescence compared to the other groups (Figure 3C), indicating that Pt-LDH can generate abundant ROS and further leading to significant toxicity Afterward, the toxicity of the HE-LDH, Pt-LDH and Ir-LDH samples was further investigated using the calcein-AM and propidium iodide (PI) double staining assays.²¹ As shown in Figure 3D, the control and comparable groups are not considerably affected by the treatments. In contrast, the Pt-LDH group showed a sharp increase in cell death, as evidenced by the strong red fluorescence intensity. These results clearly demonstrate the remarkable therapeutic effect of the Pt-LDH group in promoting cancer cell death. Such good therapeutic efficacy is mainly attributed to ROS-induced mitochondrial damage, thereby inducing cell apoptosis. Additionally, JC-1 staining was utilized to investigate the mitochondrial integrity; mitochondrial dysfunction is related to the ROS.²² It should be noted that, green JC-1 monomers show sick low-potential depolarized mitochondria, whereas red JC-1 aggregates demonstrated healthy, high-potential polarized mitochondria.¹⁸ In contrast to the control groups, as revealed in Figure 3E, Pt-LDH group was totally changed from red aggregates to green monomers in comparison to the Ir-LDH group, showing that numerous mitochondria were destroyed. Meanwhile, we have performed a concentration-dependent glucose degradation assay using a standard glucose assay kit. The experimental results demonstrate that the glucose concentration in the Pt-LDH treated group decreased over time compared to the control and Ir-LDH groups (Figure S14). Together, the above results demonstrated that this Pt-LDH can strongly disrupt homeostasis, inducing mitochondrial dysfunction and further induce cell death.

Figure 3.

In vitro experiments of Pt-LDH

(A) Schematic illustration of in vitro cellular therapy.

(B) The cytotoxicity assessed by CCK-8 under different conditions of HE-LDH, Ir-LDH, and Pt-LDH in 4T1 cells (Mean ± SD, n = 3).

(C–E) Confocal microscopy images and quantitative analysis of 4T1 cell stained with 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (ROS generation), calcein-AM/PI (red, dead cells; green, living cells), and JC-1 (red, healthy mitochondria; green, depolarized mitochondria) (Mean ± SD, n = 3). Statistical significance was evaluated using student’s t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bars, (C) 50 μm, (D) 100 μm, (E) 50 μm.

In vivo therapeutic performance of Pt-LDH

Given the promising in vitro results, we evaluated the in vivo therapeutic potential of Pt-LDH. First of all, the in vivo tumor internalization and accumulation ability of Pt-LDH sample were evaluated via fluorescence imaging. As shown in Figures 4B and 4C, Indocyanine green (ICG)-labeled Pt-LDH were progressively accumulated at the tumor site after intravenous injection, and reaching a maximum at 6 h. ICG-labeled Pt-LDH in the main organs decreased gradually over time. Notably, no obvious fluorescence intensity can be detected at 48 h post injection, demonstrating that Pt-LDH could be excreted nearly completely, making it a highly efficient in vivo therapeutic agent. Inspired by the magnetism performance on the layers of HE-LDH, Pt-LDH can be regarded as the magnetic resonance imaging (MRI) agent. The relaxation efficiency was determined to be 8.08 mM⁻¹ s⁻¹, which is higher than the clinical Gd agent²³ (Figures 4D and 4E).

Figure 4.

In vivo therapeutic performance of Pt-LDH

(A) Schematic illustration for the in vivo treatments. In vivo fluorescence imaging (B) and corresponding fluorescence intensity (C) of mice after injection of ICG-labeled Pt-LDH.

(D) Δ1/T1 vs. Fe concentration MR images for Pt-LDH in different concentration (inset is T1-weighted MR images of Pt-LDH).

(E) In vivo T1-MRI imaging of 4T1-bearing mice before and after injected with a Pt-LDH dispersion. Relative body weight (F) and tumor growth (G) of 4T1-bearing mice after different treatment (Mean ± SD, n = 5).

(H) Digital photos of isolated tumor tissue of different experimental groups.

(I) The ROS, HIF-α, Ki67, and H&E staining of tumor after different treatments. Scale bars, (I) 100 μm.

Next, the in vivo antitumor performance of Pt-LDH was further investigated (Figure 4A). The Balb/c mice were randomly assigned into three groups (n = 5 per group): (1) control group; (2) Ir-LDH group; and (3) Pt-LDH group. All samples were administered intravenously at a dose of 10 mg/kg of mouse body weight. During the treatment period, tumor volumes and body weights were recorded every two days. As expected, no noticeable body weight variations can be found in all groups, indicating the good biocompatibility of the samples (Figure 4F). Meanwhile, the results of hematoxylin and eosin (H&E) staining²⁴ of the major organs (heart, liver, spleen, lungs, and kidneys) revealed no obvious pathological damage or inflammation, confirming the desirable histocompatibility for Pt-LDH and Ir-LDH, respectively (Figure S15). According to the tumor growth curve during the therapy period of time (Figures 4G and 4H), the tumors in the control group grew quickly. Compared with the Ir-LDH-treated groups, the tumor growth of mice in the Pt-LDH group was evidently inhibited. The outstanding tumor suppression effect is ascribed to the synergistic impact of metabolic disruption and ROS generation. The above data can be further evaluated by DCFH-DA staining, hypoxia-inducible factor 1-alpha (HIF-1α),²⁵ Ki-67 staining²⁶ and H&E staining (Figure 4I) of tumor sections from each therapy. The DCFH-DA staining results of tumor sections showed intense green fluorescence in the Pt-LDH group, indicating the generation of a large amount of ROS within the tumor, while HIF-1α stained tumor sections confirmed that Pt-LDH could reverse tumor hypoxia, further verified the outstanding CAT-like activity. Additionally, Ki-67 staining showed a marked reduction in proliferating cells in the Pt-LDH group compared to the control groups, confirming effective suppression of tumor proliferation. Our results not only promise the great potential of Pt-LDH as a nanozyme agent used for cancer therapy, but may also start more adventures on LDH to benefit the biomedical applications. In summary, we have successfully constructed a high-entropy-based nanozyme, Pt-LDH. The incorporation of multiple transition metals within the LDH layer, coupled with the anchoring of Pt nanoclusters, endows the material with good multi-enzyme mimetic activities. Systematic evaluations confirm that Pt-LDH effectively alleviates tumor hypoxia, disrupts cellular redox homeostasis through ROS burst and NADH depletion, and ultimately induces mitochondrial dysfunction. Both in vitro and in vivo studies demonstrate superior tumor suppression with minimal systemic toxicity, highlighting its potential for safe and efficient anticancer application. This work not only presents a robust nanoplatform for self-enhanced cascade cancer therapy but also validates high-entropy engineering as a powerful strategy for designing multifunctional nanozymes with programmable catalytic functions.

Limitations of the study

Despite the potent synergistic anti-tumor efficacy of the Pt-LDH nanozyme, a few limitations warrant future investigation. The inherent complexity of the high-entropy matrix makes it challenging to define the exact atomic-level contribution of each transition metal to the catalytic cascade, which will require advanced in situ characterizations and theoretical calculations to fully elucidate. Furthermore, while preliminary evaluations suggest acceptable short-term safety, the long-term biological fate and chronic toxicity of this multimetallic platform—particularly concerning elements like Co, Ni, and Pt—demand comprehensive pharmacokinetic studies.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shanyue Guan (guanshanyue@mail.ipc.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data supporting the findings of this study are found within the article and its supplemental information.
• •This study does not generate any new custom code.
• •Any additional information required to reanalyze the data reported will be shared by the lead contact upon request.

Acknowledgments

This study was provided by the Beijing Natural Science Foundation (no. 7244312), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (grant no. 2019027), the National Natural Science Foundation of China (grants nos. 21805293 and 82204922), the Cross-Innovation Open Project of Food Flavor and Health at Beijing Technology and Business University (no. FFHCI-2025076), China Academy of Traditional Chinese Medicine Outstanding Young Scientific and Technological Talents Cultivation Special Programme (ZZ18-YQ-022). All animal procedures were conducted in accordance with the ethical guidelines of Chinese Academy of Traditional Chinese Medicine Guang’ anmen Hospital and were approved by Kangtai Medical Laboratory Service Hebei Co., LTD. Experimental Animal Ethics Committee (IACUC issue no. MDL2024-06-04-01).

Author contributions

M.X., Y.W., and F.Z. contributed equally to this work. M.X., Y.W., and F.Z. was responsible for study writing, study design, computational work, and data curation. Y.J. and P.J. performed calculations, statistical analysis, study writing. W.M. contributed to result analysis. S.G., S.D., and J.L. was responsible for study design and supervised study writing and revision.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Critical commercial assays
Cell Counting Kit-8	DOJINDO LABORATORIES	Cat# CK04
ROS Assay Kit -Highly Sensitive DCFH-DA	DOJINDO LABORATORIES	Cat# R252
Calcein-AM/PI Double Staining Kit	DOJINDO LABORATORIES	Cat# C542
JC-1 MitoMP Detection Kit	DOJINDO LABORATORIES	Cat# MT09
Recombinant Anti-HIF-1 alpha antibody	Servicebio	GB154936-100; RRID: AB_3094483
Anti-Ki67 Mouse mAb	Servicebio	Cat# GB121141-50
Hematoxylin-Eosin (H&E) High-Resolution Permanent Staining Kit	Servicebio	Cat# G1076
Hydrogen peroxide enzyme (CAT) activity detection kit	Solarbio	SKU: BC4785
Glucose oxidase (GOD) Assay Kit	Solarbio	SKU: BC0690
Experimental models: Cell lines
4th mammary tumor cell line 1	The Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.	Cell line: 1101MOU-PUMC001004
Experimental models: Organisms/strains
Balb/c mice	Chinese Academy of Traditional Chinese Medicine Guang’ anmen Hospital	IACUC: MDL2024-06-04-01
Software and algorithms
Fiji	ImageJ	https://imagej.net/downloads
GraphPad Prism	GraphPad	Prism 9
Origin 2021	Origin	https://www.originlab.com/
Chemicals, peptides, and recombinant proteins
NaOH	Aladdin	CAS: 1310-73-2
NaNO3	Aladdin	CAS: 7631-99-4
KOH	Aladdin	CAS: 1310-58-3
Fe(NO3)3·9H2O	Aladdin	CAS: 7782-61-8
Co(NO3)3·6H2O	Aladdin	CAS: 10026-22-9
Ni(NO3)2·6H2O	Aladdin	CAS: 13478-00-7
Cu(NO3)2·3H2O	Aladdin	CAS: 10031-43-3
MnCl2·4H2O	Aladdin	CAS: 13446-34-9
NaBH4	Aladdin	CAS: 16940-66-2
H2PtCl6·6H2O	Aladdin	CAS: 18497-13-7
H2IrCl6	Aladdin	CAS:110802-84-1
3,5,3′,5′-Tetramethylbenzidine	Aladdin	CAS: 54827-17-7
Dimethyl sulfoxide	Aladdin	CAS: 67-68-5
ICG Maleimide	Aladdin	CAS: 2143933-81-5
5,5-Dimethyl-1-pyrroline N-oxide	Aladdin	CAS: 3317-61-1
2,2,6,6-Tetramethylpiperidine	Aladdin	CAS: 768-66-1
Other
Temperature-variable electron paramagnetic resonance spectrometer	Bruker	E500
Atomic Force Microscope	Bruker	Dimension Icon
X-ray photoelectron spectrometer	Thermo Fisher Scientific of the United Kingdom	ESCALAB 250xi
Confocal imaging microscope	Nikon of Japan	ARsiMP-LSM-Kit-Legend Elite-USX
Ultraviolet-visible spectrophotometer	Hitachi of Japan	U‒3010
Enzyme Labeling Instrument	Thermo Fisher Scientific of the United Kingdom	Multiskan FC
Small animal live imaging system	PerkinElmer	IVIS SPECTRUM
X-ray diffractometer	Bruker	D8 focus

Method details

Materials

Iron nitrate monohydrate (Fe(NO₃)₃·9H₂O), Cobalt nitrate hexahydrate (Co(NO₃)₃·6H₂O), Nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O), Manganese chloride tetrahydrate (MnCl₂·4H₂O), Sodium borohydride (NaBH₄), Hexahydrate of chloroplatinic acid (H₂PtCl₆·6H₂O), Hydrate of chloroiridic acid (H₂IrCl₆) and methyl blue (MB) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Copper nitrate solution Cu(NO₃)₂⋅3H₂O, sodium hydroxide (NaOH), sodium carbonate anhydrous (NaCO₃), 1,3-Diphenylisobenzofuran (DPBF), Reduced Coenzyme I Dihydrate (NADH), and 3,3,5,5-tetramethylbenzidine (TMB), and indocyanine green dye (ICG) were purchased from Sigma-Aldrich. Calcein acetoxymethyl ester (Calcein-AM), propidium iodide (PI), 2,7 Dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich Co. Ltd (St. Louis, MO, USA). Dulbecco’s modified Eagles medium (DMEM), 0.25% trypsin-EDTA, Phosphate buffered saline (PBS), and Fetal bovine serum (FBS) were purchased from Beijing Solarbio Science and Technology Company. The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and cell counting Kit 8 (CCK-8) were purchased from Dojindo China Co., Ltd. All of the cells were obtained from the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. All other reagents and solvents were of analytical purity and used without further purification. The DI water was used during all experiments.

Characterization

Powder X-ray diffraction (XRD) patterns were collected on a Bruker X-ray diffractometer (D8 focus, Cu Kα, λ = 0.15178 nm) operated at a scan rate of 0.1°/s. The TEM (JEOL-2100) instrument was equipped with an energy-dispersive X-ray spectroscopy (EDS) analyzer for quantitative elemental analysis. Atomic force microscopy (AFM, Veeco) was employed to measure the dimensions and thickness. XPS data was acquired on an ESCALAB 250Xi (Thermo Scientific), equipped with a non-monochromatized Al-Kα X-ray source (hν = 1486.7 eV). Zeta potentials were determined on NanoBrook 90Plus Zeta. UV-Vis absorption spectra were acquired using a Hitachi U-3010 spectrophotometer. Elemental composition was analyzed via inductively coupled plasma atomic emission spectroscopy (ICP-AES, 710-OES), and electron spin resonance (ESR) spectroscopy was conducted at 110 K with a JES-FA200 instrument at 110 K or a Bruker EMX PLUS at room temperature (300 K). A Thermo Multiskan FC was used to investigate cell viability. Confocal fluorescence images were obtained on a Nikon A1R Eclipse Ti confocal laser scanning microscope fitted with a 40× water immersible objective.

Synthesis of HE–LDH

HE–LDH with a molar ratio of 1:1:1:1:1 was synthesized via a co-precipitation method. Solution I was prepared by dissolving 0.004 mol g of MnCl₂·4H₂O, 0.004 mol of Co(NO₃)₂·6H₂O, 0.004 mol of Ni(NO₃)₂·6H₂O, 0.004 mol of Cu(NO₃)₂·3H₂O, and 0.004 mol of Fe(NO₃)₃·9H₂O in 100 mL of deionized water. Solution II was obtained by dissolving 0.05 mol of NaOH and 0.02 mol of NaNO₃ in 100 mL of deionized water. The two solutions were loaded into channels A and B of a syringe pump, respectively. A flask equipped with a mechanical stirrer was placed in a water bath maintained at 80 °C. Solutions I and II were added dropwise into the flask under vigorous stirring, while the pH of the mixture was kept between 8.5 and 9.5. After complete addition, the reaction proceeded for 6 h under continuous stirring. The resulting precipitate was collected by centrifugation at 3500 rpm, washed twice with deionized water and ethanol, and then freeze-dried for 12 h to obtain the final HE–LDH product.

Synthesis of Ir–LDH

Ir–LDH was prepared by the NaBH₄ reduction method. First, 1 g of the HE-LDH synthesized was added to 100 mL of deionized water and stirred for 1 h to form a uniform dispersion. Then, 2 mol of H₂IrCl₆ was added and stirred for another 0.5 h. Next, 0.04 mol of KOH was dissolved in 20 mL of deionized water, and 0.01 mol of NaBH₄ was dissolved in 20 mL of deionized water (with two drops of KOH solution added during the process to maintain an alkaline environment). Under vigorous stirring, the NaBH₄ solution was slowly dropped into the mixed dispersion and stirred for 6 h. The mixture was then centrifuged at 3500 rpm, and the precipitate was washed twice with deionized water and ethanol. The obtained precipitate was dried in a freeze dryer for 12 h to prepare Ir–LDH.

Synthesis of Pt–LDH

Pt–LDH was prepared via a NaBH₄ reduction method. Specifically, 1 g of as-synthesized HE-LDH was dispersed in 100 mL of deionized water and stirred for 1 h to form a homogeneous suspension. Then, 0.002 mol of H₂PtCl₆·6H₂O was added, and stirring was continued for another 0.5 h. Separately, 0.04 mol of KOH was dissolved in 20 mL of deionized water, and 0.01 mol of NaBH₄ was dissolved in another 20 mL of deionized water. Two drops of the KOH solution were added to the NaBH₄ solution to maintain alkaline conditions. The NaBH₄ solution was then slowly introduced into the Pt-containing mixture under vigorous stirring. The reaction was allowed to proceed for 6 h under continuous stirring. The resulting product was collected by centrifugation at 3500 rpm, washed twice with deionized water and ethanol, and finally freeze-dried for 12 h to obtain Pt–LDH.

Detection of ROS generation

To verify the production of reactive oxygen species (ROS), TMB was used as a probe. The sample and H₂O₂ were added to a PBS buffer (pH = 6.5) containing TMB. The absorbance of the reaction mixture at 652 nm was recorded every minute using a UV-Vis spectrophotometer to monitor ROS generation catalyzed by different samples in the presence of H₂O₂. For the detection of singlet oxygen (¹O₂), DPBF was employed as a probe. The decrease in absorbance at 450 nm was measured at 1-min intervals under the same conditions to evaluate ¹O₂ production. The peroxidase-like (POD) activity of the samples was further investigated via H₂O₂ kinetics with different concentration. Michaelis–Menten parameters were determined by monitoring the absorbance at 652 nm over time after the addition of TMB under aerobic conditions. The kinetic parameters, including the Michaelis constant (K_m) and the maximum reaction rate (V_max), were obtained by fitting the data to the Michaelis–Menten equation:

$$V=V_{\mathit{max}}\left[S\right]/K_m+\left[S\right]$$

where V represents the initial velocity, V_max is the maximum reaction rate, and [S] denotes the substrate concentration.

Detection of GOx-like activity

To evaluate the GOx-like activity of Pt–LDH, the generation of product gluconic acid during GOx-like reaction was detected using a gluconic acid-specific colorimetric assay in the presence of hydroxylamine and FeCl₃. The generated gluconic acid can react with hydroxylamine and subsequently form complex with Fe³⁺, which would lead to a red complex with a major absorbance at 505 nm. Specifically, 1 mL of aqueous solution containing EDTA (5.0 mM) and triethylamine (0.15 mM), as well as 100 μL of aqueous solution containing NH₂OH (3.0 M) were added to 100 μL of the GOx-like catalytic reaction solution. After 25 min of incubation, 500 μL of aqueous solution containing HCl (1.0 M), FeCl₃ (0.1 M), and CCl₃COOH (0.25 M) was added to the above aqueous solution, and the absorbance of the mixture was recorded using a UV-vis spectrophotometer.

Catalytic reactions of Pt–LDH with NADH

To evaluate NADH consumption, 200 μL of NADH (1 mg/mL) was added to 1.8 mL of the sample solution (20 μg/mL). The mixture was reacted for 30 min, and the absorbance of the supernatant at 260 nm and 340 nm was measured every 5 min using a UV-Vis spectrophotometer. To further investigate NADH oxidation, 800 μL of NADH (5 mM) was mixed with 1 mL of the sample solution (1 mg/mL) and incubated for 5 min. The absorbance at 563 nm was monitored by UV-Vis spectroscopy to assess the promotion of NADH oxidation.

Electron spin resonance (ESR) spectroscopy

ESR measurements were conducted to detect superoxide anion radicals (·O₂⁻), singlet oxygen (¹O₂) and hydroxyl radical (·OH) using DMPO as the spin trap for ·O₂⁻ in DMSO solution, TEMP for ¹O₂, and DMPO for ·OH. The corresponding ESR signals generated by different samples were recorded and analyzed.

Cytotoxicity assay

4T1 cells were cultured in flasks under a 5% CO₂ atmosphere at 37 °C. The cells were then seeded into a 96-well plate at a density of 1×10⁴ cells per well and incubated for 24 h. Afterward, the cells were treated with various concentrations of the samples under different tumor microenvironment-mimicking conditions and further incubated for 24 h. A mixture of CCK-8 and culture medium was added to each well, followed by incubation for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated based on the relative absorbance values.

Analysis of ROS generation

The production of ROS in cells was evaluated using DCFH-DA as a fluorescent probe, which is oxidized by ROS to generate fluorescent DCF. Briefly, 4T1 cells were seeded into culture dishes at a density of 2 × 10⁵ cells per dish and incubated for 24 h. The culture medium was then replaced with 1 mL of fresh medium containing different samples. After 2 h of incubation, the cells were washed twice with PBS and incubated with 1 mL of PBS containing DCFH-DA at 37 °C for 30 min. After removing excess probe by washing, fluorescence images were acquired using confocal laser scanning microscopy (CLSM) with an excitation wavelength of 488 nm.

Live/dead cell assay

To visualize cell viability, treated cells were double-stained with Calcein-AM and propidium iodide (PI) under different conditions. After 30 min of incubation, the cells were washed once with PBS and imaged in fresh PBS using CLSM. Calcein-AM, taken up by live cells, produces green fluorescence under 488 nm excitation, whereas PI, which enters dead cells, emits red fluorescence when excited at 561 nm.

In vivo fluorescence imaging

The biodistribution of the samples in mice was investigated using a small animal in vivo imaging system. The samples were mixed and stirred with the near-infrared dye ICG for 8 h, then centrifuged and redispersed in PBS to obtain ICG-labeled samples. When the tumor volume exceeded 100 mm³, the ICG-labeled samples were administered to nude mice via tail vein injection. Fluorescence images were acquired at 0, 1, 3, 6, 12, 24, and 48 h post-injection to monitor the in vivo distribution of the samples.

Therapeutic efficacy study

Mice were randomly divided into groups (n = 5 per group) and administered different samples via injection. Throughout the treatment period, the agents were administered every two days. Tumor volumes were measured using a vernier caliper, and body weights were recorded every two days for a total of 14 days. Tumor volume was calculated using the formula:

$$TumorVolume=0.5\times length\times {\left(width\right)}^2$$

After 14 days of treatment, the mice were euthanized, and major organs along with tumor tissues were collected through dissection. The tissues were fixed in 4% formaldehyde solution for 24 h, followed by sectioning and staining using an H&E staining kit. Histopathological changes in various organs and tissues were examined under a CLSM.

Quantification and statistical analysis

The mean value and standard deviation (mean ± SD) of all data showed that the statistical analysis ∗p < 0.05 of different groups through Graphpad Prism 9. was considered statistically significant, ∗∗p < 0.01 and ∗∗∗p < 0.001 were considered notably significant. Exact p-values, statistical tests used, and the number of replicates (n) are reported in the corresponding figure legends.