Functional nanozyme system for synergistic tumor immunotherapy via cuproptosis and ferroptosis activation

Abstract

Elevated copper levels induce tumor cuproptosis and ferroptosis, leading to immunogenic cell death and subsequent antitumor immune responses. However, dysregulated copper metabolism in tumor cells maintains homeostatic copper balance, while hypoxic microenvironments hinder therapeutic efficacy. In this study, we present a nanozyme system, termed CussOMEp, comprising a copper-based nanovector (CussNV) that is PEGylated and loaded with omeprazole, a copper transporter inhibitor, to enhance tumor synergistic immunotherapy by promoting cuproptosis and ferroptosis. CussNV is assembled from dithiodiglycolic acid and copper ions, exhibiting peroxidase, glutathione oxidase, and catalase-like activities, along with responsive degradability. This nanozyme alleviates tumor hypoxia by producing oxygen, induces ferroptosis through the generation of lethal hydroxyl radicals, and depletes glutathione. Additionally, omeprazole increases cellular copper concentration and oxidative stress by inhibiting the intracellular copper-transporting ATPase 1 (ATP7A), enhancing lipoylated protein oligomerization and cuproptosis. In a breast tumor mouse model, CussOMEp elicits robust antitumor immune responses, including dendritic cell maturation and T cell proliferation. When combined with PD-1 antibodies (αPD-1), CussOMEp significantly inhibits tumor metastasis in bilateral and lung metastatic models. This work presents a functional nanozyme system as a promising strategy for synergistic tumor immunotherapy leveraging ferroptosis and cuproptosis

Graphical abstract

Introduction

The emerging roles of metal ions, particularly copper, in cancer biology have garnered considerable attention due to their dual capacity to promote tumorigenesis and activate cell death pathways [1,2,3]. Elevated copper levels within tumors can initiate distinct forms of cell death, including cuproptosis and ferroptosis [4, 5]. Cuproptosis, a recently identified form of cell death, arises from copper-induced proteotoxic stress, leading to the aggregation of lipoylated proteins within the tricarboxylic acid cycle and subsequent loss of iron-sulfur cluster proteins [6]. In contrast, ferroptosis, which is characterized by lipid peroxidation and triggered by oxidative stress, has been recognized as a critical mechanism of cell death across various malignancies [7,8,9,10,11,12,13,14]. Notably, copper can also induce ferroptosis by increasing reactive oxygen species (ROS) levels [15,16,17,18,19,20,21,22]. Mechanistically, both cuproptosis and ferroptosis are linked to immunogenic responses that may enhance antitumor immunity [23,24,25,26,27,28,29,30,31].

However, the therapeutic potential of these pathways is impeded by dysregulated copper metabolism in tumor cells, which often maintain a homeostatic balance that limits copper accumulation and its cytotoxic effects [32]. Tumor cells require higher concentrations of copper for their aberrant proliferation compared to normal cells [33, 34]. Consequently, they exhibit elevated expression of copper uptake transporters to meet their increased demand for copper, while simultaneously expressing high levels of copper exporters, such as copper-transporting ATPase 1 (ATP7A), to sustain biological homeostasis [35,36,37]. ATP7A is specifically responsible for the transport of copper ions into tumor cells [38]. Under conditions of low copper availability, ATP7A localizes to the Golgi membrane network. As copper levels increase, ATP7A translocate to the plasma membrane or intracellular vesicles, facilitating copper transport to post-Golgi vesicles [39]. Subsequently, these copper-laden vesicles fuse with the plasma membrane, releasing copper into the extracellular environment. Therefore, the elevation of copper levels through the use of ionophores or nanoparticles is counteracted by ATP7A activity. Inhibiting ATP7A expression may reduce the copper loss induced by copper-containing ionophores or nanoparticles, thereby enhancing therapeutic efficacy.

Additionally, the elevated levels of glutathione (GSH) in tumor cells and their microenvironment impede both ferroptosis and cuproptosis [40, 41]. As a reducing agent, GSH diminishes cellular ROS levels and acts as a cofactor for glutathione peroxidase 4 (GPX4), a key regulator of ferroptosis, thereby undermining this form of cell death [42]. Furthermore, GSH functions as a copper chelator, inhibiting cuproptosis [43, 44]. More critically, the hypoxic microenvironment characteristic of solid tumors further reduces the efficacy of therapies targeting ferroptosis and cuproptosis pathways, while also promoting a suppressive immune environment [45,46,47]. To address these challenges, innovative strategies are necessary to harness the cytotoxic potential of copper while mitigating the constraints imposed by tumor microenvironments.

In this study, we present a novel functional nanozyme system, CussOMEp, which integrates a copper-based nanovector (CussNV) with the copper transporter inhibitor omeprazole (OME). CussNV exhibits peroxidase (POD), catalase (CAT), and GSH peroxidase (GSHox)-like activities, enabling the conversion of tumor-derived hydrogen peroxide (H₂O₂) into cytotoxic hydroxyl radicals (^•OH) and oxygen while concurrently depleting GSH. Furthermore, CussOMEp displays pH- and GSH-responsive biodegradability within the tumor microenvironment, facilitating the release of OME, which inhibits ATP synthase (ATP7A) and enhances therapeutic efficacy. By leveraging the unique properties of CussOMEp, we aim to improve tumor immunotherapy through the induction of cuproptosis and ferroptosis. This strategy is designed not only to increase copper ion levels and oxidative stress within tumor cells but also to modulate the tumor microenvironment, thereby enhancing antitumor immune responses and reducing metastatic potential. When combined with PD-1 antibodies (αPD-1), CussOMEp demonstrates significant therapeutic efficacy in models of bilateral and lung metastases (Scheme 1). Our findings highlight the promise of functional nanozyme systems as a transformative approach in cancer therapy, addressing the metabolic and microenvironmental barriers that currently limit treatment effectiveness.

Scheme 1

Fabrication processes of CussOMEp nanozyme system and its mechanisms in tumor therapy

Materials and methods

Chemicals and reagents

N, N-dimethylformamide, dithiodiglycolic acid, copric chloride dihydrate, triethylamine, indocyanine green (ICG), omeprazole, 3,3’,5,5’-tetramethylbenzidine (TMB), methylene blue (MB), 1,2-diaminobenzene (OPD), and triethylamine (TEA) were bought form Shanghai Aladdin Biochemical Technology Co., LTD. Ethanol was acquired from China National Pharmaceutical Group Corporation. Polyvinyl pyrrolidone (PVP, molecular weight = 40000) was purchased from Sigma-Aldrich. DSPE-mPEG (molecular weight = 2000) was provided by Shanghai Yare Co., LTD. Rhodamine B hydrazide (RBH) was bought form Biofount Technology Co., LTD. Dulbecco’s modified eagle medium (DMEM) and phosphate buffer solution (PBS) were obtained from Service Biotechnology Co., LTD. Fetal bovine serum (FBS) was purchased from Hyclone. Ru(dpp)₃Cl₂, monobromobimane, and D-luciferin potassium salt were obtained from Macklin Biochemical Technology Co., LTD. BBoxiProbe O26 was obtained from Bestbio Technology Co., LTD. 5, 5’-dithio-bis (2-nitrobenzoic acid) (DTNB), 4′,6-diamidino-2-phenylindole (DAPI), and anti-HSP70 (Cat# AF1156) were obtained from Beyotime Biotechnology. HRP-conjugated affinipure goat anti-mouse IgG (H + L) (Cat# 15014), HRP-conjugated affinipure goat anti-Rabbit IgG(H + L) (Cat# 15015), anti-β-actin (Cat# 66009-1-Ig), anti-DLAT (Cat# 13426-1-AP), and anti-FDX1 (Cat# 12592-1-AP) were gained from Proteintech. ATP7A (Cat# E-AB-13081) antibodies were obtained from Elabscience Biotechnology Co., LTD. Annexin V-FITC/PI apoptosis kit and CCK8 kit were obtained from Yeasen Biotechnology Co., LTD. Anti-mouse PD-1 (Cat# BE0273-100MG) was acquired from Univ Company. India ink was bought from Shanghai Shifeng Biotechnology Co., LTD. All chemicals were used without further purification. All antibodies used in flow cytometry are listed in Table S1.

Synthesis of CussNV

To begin, 300 mg of PVP was dissolved in 3 mL of DMF and subjected to ultrasonic agitation for 5 min. Next, 5.2 mg of 2,2’-disulfanediyldiacetic acid and 10 mg of CuCl₂·2 H₂O were sequentially added to the solution, which was stirred for an additional 10 min. Subsequently, 0.6 mL of TEA was rapidly introduced under vigorous stirring, followed by the swift addition of a mixture of 6.25 mL of DMF and 3.75 mL of ethanol. The resulting solution was transferred into a hydrothermal synthesis reactor preheated to 150 °C and maintained at this temperature for 12 h. After cooling to room temperature, the products were collected by centrifugation at 10,000 rpm for 10 min (Sorvall ST 16R centrifuge, Thermo Fisher) and washed three times with ultrapure water.

Synthesis of pegylated CussOMEp

To synthesize CussOMEp, 400 µg of CussNV was dispersed in 1 mL of ultrapure water containing 0.4 µmol of OME and gently stirred overnight to obtain CussOME. The resulting CussOME nanoparticles were collected by centrifugation at 10,000 rpm for 10 min and washed three times with ultrapure water. For PEGylation, 200 µg of DSPE-mPEG was added to the CussOME solution and allowed to react for 6 h. The PEGylated nanoparticles (CussOMEp) were then collected by centrifugation at 10,000 rpm for 10 min and washed three times with ultrapure water.

Characterization of Cussomep

Transmission electron microscopy (TEM) images were carried out on a Hitachi HT7700 transmission electron microscope. Atomic force microscope (AFM) images were conducted on an atomic force microscope (BRUKER Dimension Icon). X-ray diffraction (XRD) was conducted by a Rigaku Miniflex600 X-ray diffractometer. BET results were obtained from ASAP2020 of micromeritics (Version 4.03.). X-ray photoelectron spectroscopy (XPS) spectra were recorded by an X-ray photoelectron spectrometer (Thermo SCIENTIFIC Nexsa, Thermo Fisher). The mapping images were acquired form a transmission electron microscope (JEM 2100 F, JEOL). The zeta potential and hydrodynamic size were measured by a particle and molecular charge analyzer (Zetasizer Nano ZS, Malvern). FTIR spectra were recorded by a Fourier transform infrared spectrometer (Thermo Nicolet IS 50, Thermo Fisher).

Degradation behaviors of cussomep

CussNV nanoparticles were dissolved in PBS at 6.0 and 7.4 pH with or without 10 mM GSH for 10 h, the morphology of CussNV was then characterized using a TEM.

Release behaviors of OME and copper ions from CussOMEp

10 mg of CussOMEp were dispersed in PBS solutions at pH 7.4 and pH 6.0 with or without 10 mM GSH. At specified time points, the released OME and cupric in the supernatant were collected by centrifugation at 10,000 rpm for 20 min and the copper contents and OME were quantified using atomic absorption spectrometry and UV-visible spectrophotometer (UV-8000 S, Shanghai Metash Instruments Co., Ltd.), respectively. The absorption peak intensity at 278 nm of OME was used to determine the standard curve.

Dissolved oxygen determination

A solution containing 50 µg/mL CussNV and 10 mM H₂O₂ was prepared, and its oxygen content was measured using a dissolved oxygen analyzer (JPB607A, Leici).

^•OH generation ability of CussNV

To evaluate the ^•OH generation capacity of CussNV, a 50 µg/mL solution of CussNV was dispersed in 1 mL of PBS and treated with 0.5 mM TMB, 50 µM MB, or 20 mM OPD, followed by the addition of 100 µM H₂O₂. The mixture was incubated for 30 min, after which the absorbance of the resulting oxidation products was measured using a UV-visible spectrophotometer and a microplate reader (1510, Thermo Fisher). Additionally, electron spin resonance (ESR) spectroscopy was performed to verify the generation of hydroxyl radicals (^•OH). Briefly, 50 mM H₂O₂ and 100 µg/mL CussNV were mixed in a buffer at pH 6.0 for 15 min. Subsequently, 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added, and the ESR spectra were recorded to detect ^•OH production. A sample without CussNV served as the control group.

For the analysis of POD-like activity, the 250 µg/mL CussNV were thoroughly combined with TMB solution, followed by the addition of H₂O₂ at concentrations of 2.5 mM, 5 mM, 10 mM, 15 mM, and 20 mM. The absorbance at 655 nm was measured within 30 min to determine the kinetic parameters of the activity of the POD-like enzyme.

GSH depletion ability of CussNV

The GSH depletion capacity of CussNV was assessed using the DTNB reagent. A solution of PBS containing 10 mM GSH was incubated with varying concentrations of CussOMEp at 37 °C for 12 h. Following incubation, the mixture was reacted with DTNB for 4 h. The ultraviolet absorbance of the above solution at 412 nm was then measured using a UV-visible spectrophotometer.

For assessment the GSHox-like activity, CussNV at a concentration of 250 µg/mL was incubated with GSH at concentrations of 0.1 mM, 0.75 mM, 1.25 mM, 2 mM, and 2.5 mM. Following incubation, the mixture was centrifuged, and the resulting supernatant underwent a colorimetric reaction with DTNB. The absorbance at 412 nm was subsequently measured within 1 h to determine the kinetic parameters of nanozyme.

Cell culture

Luc-4T1 cells were generated by lentiviral transfection of the luciferase reporter gene into 4T1 cells. Both 4T1 and Luc-4T1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37 °C in a 5% CO₂ atmosphere, and routinely sub-cultured.

Cell uptake

The FITC labeled CussNVp (CussNVp@FITC) was synthesized for cellular uptake investigation. Briefly, 50 µg 4T1 cells were incubated with 50 µg FITC for 12 h, then the mixture was centrifuge for several times to obtain CussNVp@FITC. After that, 4T1 cells were seeded into a 6-well plate at a density of 1.0 × 10⁵ cells per well and cultured for 24 h. Subsequently, the cells were treated with CussNVp@FITC at a concentration of 50 µg/mL for various time intervals. After treatment, the presence of green fluorescence signals within the cells were assessed using flow cytometry (Novocyte 3130, ACEA).

Cytotoxicity evaluation

4T1 cells were seeded into 96-well plates at a density of 1.0 × 10⁴ cells per well and cultured for 24 h. Cells were then treated with varying concentrations of OME (0, 1, 2, 5, 10, 20, 50, and 100 µM), CussNVp (0, 20, 40, 60, 80, 100, 150, and 200 µg/mL), or CussOMEp (0, 20, 40, 60, 80, 100, 150, and 200 µg/mL) for an additional 24 h. After treatment, the media were replaced with fresh medium containing 10 µL of CCK-8 solution per well. Cell viability was determined by measuring absorbance at 450 nm.

Intracellular Cu²⁺ detection

4T1 cells (1 × 10⁶) were seeded into confocal dishes and allowed to adhere. After 24 h of treatment with various probes, the cells were incubated with a Cu²⁺-specific fluorescence probe (RBH, 1 µM) at 37 °C for 30 min, followed by fixation with 4% paraformaldehyde for 15 min. The cells were then stained with DAPI for 10 min. Fluorescence imaging was conducted using a confocal laser scanning microscopy (CLSM, FV3000, Olympus), with excitation at 510 nm and emission at 578 nm for the Cu²⁺ probe, and excitation at 360 nm and emission at 460 nm for DAPI. In addition, the intracellular Cu²⁺ concentration was quantified by using the Cu²⁺ fluorescence probe and recording using a fluorescence spectrophotometer (F-7100, Hitachi) with excitation at 510 nm and emission at 578 nm. The statistical results of fluorescent images were counted using the Image J software.

Intracellular distribution of ATP7A

4T1 cells (1 × 10⁶) were seeded into confocal dishes and incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. The cells were then fixed in 4% paraformaldehyde for 15 min and blocked with 5% BSA for 2 h. Next, the cells were incubated overnight with ATP7A antibody (1: 200), followed by a 1-hour incubation with goat anti-rabbit IgG(H + L) conjugated to AF488 at 4 °C. After staining DAPI for 10 min, the fluorescence intensity of the cell samples was imaged using a CLSM. Fluorescence excitation and emission wavelengths were set at 488 nm and emission at 520 nm for the AF488-conjugated antibody, and at 360 nm and 460 nm for DAPI, respectively.

Intracellular oxygen, ^•OH and liperfluo evaluation

4T1 cells (1 × 10⁶) were seeded into confocal dishes and incubated for 24 h to allow for adherence. After that, cell samples were incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. Then the cells were then treated with either 50 µM of Ru(ddp)₃Cl₂, a fluorescent oxygen probe, at 37 °C for 2 h, or a 1: 1000 dilution of BBoxiProbe O26, a ^•OH fluorescent probe, at 37 °C for 30 min or 10 µg/mL liperfluo, a ferroptosis fluorescent probe, at 37 ℃ for 30 min. After treatments, cells were fixed with 4% paraformaldehyde at 4 °C for 15 min. Following fixation, the cells were stained with DAPI for 10 min and imaged using a CLSM. The imaging parameters were set as follows: excitation at 488 nm and emission at 510 nm for both Ru(ddp)₃Cl₂, BBoxiProbe O26, and liperfluo, and excitation at 360 nm and emission at 460 nm for DAPI.

Intracellular GSH detection

Nonfluorescent bromobimane can be converted into fluorescent compounds in the presence of small thiols, such as GSH. To assess intracellular GSH levels, 4T1 cells were received with indicated treatments and stained with 100 µM bromobimane at 37 °C for 30 min. Blue fluorescence was detected using a CLSM, with excitation at 392 nm and emission at 478 nm, to visualize GSH levels.

Western blot analysis

4T1 cells were seeded into a 6-well plate at a density of 1.0 × 10⁵ cells per well and cultured for 24 h. The cells were then treated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. After treatment, the cells were harvested, washed with ice-cold PBS, and lysed. β-actin served as the loading control, detected using anti-actin antibodies, while the expression levels of GPX4, HSP70, DLAT, and FDX1 were assessed using their respective antibodies (dilution 1: 1000). Membranes were then incubated with secondary antibodies against rabbit IgG or mouse IgG (dilution 1: 5000) and imaged using a multifunctional fluorescent and luminescent gel imager (Cheml XRQ, Gene Company Limited).

Apoptosis analysis

4T1 cells (1.0 × 10⁶) were seeded into 6-well plates and incubated for 24 h in the presence of OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL). After treatment, the cells were stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI), followed by fluorescence signal analysis using a FCM.

Hemolysis assay

All animal experiments were conducted in accordance with protocols approved by the Animal Experimental Ethics Committee of Fujian Normal University (Approval No. IACUC-20230036). Female Balb/c mice (4–6 weeks old) were obtained from Shanghai Slack Laboratory Animal Co., LTD. Blood samples were collected via orbital puncture and placed in anticoagulant tubes. Red blood cells were isolated by centrifugation at 3000 rpm for 5 min and diluted to a final concentration of 2%. Various concentrations of CussNVp and CussOMEp were incubated with the red blood cells for 2 h at 37 °C. Phosphate-buffered saline (PBS) and 0.5% Triton X-100 were used as negative and positive controls, respectively. Hemolysis was quantified by measuring the absorbance at 576 nm, and hemolysis rates were calculated using the following formula:

Hemolysis (%) = (OD576_sample - OD576_N) / (OD576_P - OD576_N) × 100%.

Biosafety evaluation

Balb/c mice (4–6 weeks old) were randomly assigned to two groups (n = 6 per group) and intravenously administered either PBS or 300 µg of CussOMEp per mouse. Body weights were measured every five days for 90 days. Blood and serum samples were collected to assess key physiological parameters. Major organs were harvested, weighed, and subjected to H&E staining for morphological analysis.

In vivo biodistribution assay

ICG-labeled CussNVp (denoted as CussICGp) was synthesized for in vivo imaging. In brief, 100 µg of ICG was mixed with 100 µg of CussNVp and incubated overnight. The unbound ICG was then removed by centrifuge, and the loading rate of ICG was determined using UV-visible spectrophotometry. Six 4T1 tumor-bearing mice were randomly divided into two groups: (1) Free ICG, and (2) CussICGp. Each group of mice received a 100 µg dosage of ICG via tail vein injection. Fluorescence imaging was conducted using an in vivo imaging system (IVIS) at various times (0, 0.5, 2, 4, 8, 12, 24, and 48 h). At 48 h post-injection, the mice were euthanized, and their tumor tissues and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo imaging. Furthermore, to quantify the tumor-targeting capacity of CussNVp, twelve 4T1 tumor-bearing mice were administered either PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously injection, and the mice were euthanasia at 48 h post-injection. The tumor tissues were collected, and the copper contents were assessed using the fluorescent probe.

In vivo synergistic antitumor efficacy

The 4T1 tumor model was established by subcutaneously injecting 1 × 10⁶ 4T1 cells into the right flank of healthy female Balb/c mice. Once tumors reached a volume of 100 mm³, the mice were randomly assigned to four groups (n = 4) and intravenously administered one of the following treatments: PBS, 0.054 µmol OME per mouse, 300 µg CussNVp per mouse, or 300 µg CussOMEp per mouse. Tumor volumes and body weights were monitored every two days.

On day 14, mice were euthanasia, the tumor tissues and tumor-draining lymph nodes were collected. Tumor tissues were subjected to H&E staining, as well as IHC analysis using anti-HIF-1α, anti-GPX4, and anti-FDX1 antibodies. Besides, single-cell suspension of tumor tissues was prepared for immune profiling. Macrophage polarization was evaluated using anti-CD45-PE/Cy5, anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, and anti-CD206-Alexa Fluor 647. Cytotoxic T lymphocytes and helper T cells were analyzed by staining with anti-CD45-PE, anti-CD3-PE/Cy7, anti-CD4-APC, and anti-CD8a-PerCPCy5.5. Neutrophil infiltration was assessed with anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, and anti-Ly6G-PE, while B cell infiltration was measured using anti-B220-FITC. In addition, the single-cell suspension of tumor-draining lymph nodes were prepared for DC maturation analysis. Single-cell suspensions were prepared, and cells were stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE antibodies at 4 °C for 30 min, followed by flow cytometric analysis.

In vivo anti-abscopal activity

Primary tumors were established by subcutaneously injecting 1 × 10⁶ 4T1 cells into the right flank of healthy female Balb/c mice. The tumor-bearing mice were then randomly divided into four groups (n = 8) and administered either PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously on day 0. Additionally, mice received αPD-1 (1 mg/kg) treatment on days 2, 5, and 8. On day 9, 2 × 10⁵ 4T1 cells were injected subcutaneously into the left flank to establish abscopal tumors. Tumor volumes and body weights were recorded every other day for up to 34 days.

On day 34, mice were euthanasia, and the lymph nodes, tumors, and spleens were collected to make single-cell suspensions for immune response analysis. For DC maturation assessment, lymph node single-cell suspensions were stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE at 4 °C for 30 min. For tumor single-cell suspensions analysis, macrophage polarization was evaluated by staining with anti-CD45-PE/Cy5, anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, anti-CD86-APC, and anti-CD206-FITC. Cytotoxic T lymphocyte and helper T cell populations were examined by staining with anti-CD3-PE/Cy7, anti-CD4-BV421, and anti-CD8a-APC/Cy7. Tumor cells were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set for 45 min prior to additional staining. After treating with brefeldin A for 6 h before staining with anti-IFNγ-APC. Regulatory T cell inhibition was evaluated using anti-Foxp3-PE following centrifugation and analyzed by flow cytometry. Memory T cells were analyzed by staining spleen cell suspensions with anti-CD3-APC, anti-CD8a-PerCP/Cy5.5, anti-CD44-FITC, and anti-CD62L-BV650.

In vivo anti-metastatic activity

To establish primary tumors, 1 × 10⁶ 4T1 cells were subcutaneously injected into the right flank of Balb/c mice. Once the tumor volume reached approximately 100 mm³, the mice were intravenously administered PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp (n = 8). Subsequently, αPD-1 antibody (1 mg/kg) was administered intravenously on days 2, 5, and 8. On day 9, the mice received an intravenous injection of 2 × 10⁵ Luc-4T1 cells to induce lung metastasis. For bioluminescence imaging, D-luciferin potassium salt (150 mg/kg) was intraperitoneally injected on days 21, 25, and 30. Imaging was performed using an IVIS Spectrum (PerkinElmer) under anesthesia 10 min after the D-luciferin injection. Tumor volumes and body weights were recorded bi-daily for up to 38 days. To assess lung metastasis, lung tissues were filled with India ink and subsequently stained with H&E.

Density functional theory simulation

The initial structure of the nanocluster was obtained through conformational search with the CREST program. After obtaining the wavefunctions based on B3LYP functional and def2-SVP basis in the Gaussian 16 program (Rev A.03), the weak interaction analysis was performed using the interaction region indicator (IRI) in the Multiwfn program [48, 49].

Statistical analysis

Quantitative data are expressed as mean ± standard deviation. Statistical analyses were performed using one-way ANOVA with GraphPad Prism 8.0 software. Statistical significance is indicated by an asterisk, with the following designations: ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. A P value of 0.05 or less was considered statistically significant.

Results and discussion

Synthesis and characterization of CussOMEp

CussNV was synthesized through the coordination of Cu²⁺ ions with disulfide bonds (S-S) in dithiodiglycolic acid. Transmission electron microscopy (TEM) images revealed that CussNV displayed an irregular morphology with a small particle size (Fig. 1a). Energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed the presence of Cu and S in the structure (Figure S1). Furthermore, we conducted density functional theory (DFT) simulations to investigate the coordination interactions between copper ions and dithiodiglycolic acid molecules in CussNV at the atomic level. As shown in Fig. 1b, Cu atoms exhibited 5- or 6-coordination with dithiodiglycolic acid molecules, primarily through carboxyl (-COOH) groups and sulfur atoms. The calculated electron density isosurface showed elevated electron densities in the vicinity of Cu atoms compared to those surrounding other atoms (Fig. 1c). This suggests an ionic nature of the coordination between Cu and -COOH or S groups. Additionally, the accumulation of electrons around the Cu atoms indicates a significant likelihood of the reduction of Cu²⁺ to Cu⁺ ions.

Brunauer-Emmett-Teller (BET) analysis demonstrated that CussNV exhibited the type IV isotherms with a pore volume of 0.159 cm³/g and a surface area of 19.23 m²/g (Fig. 1d). Besides, the CussNV exhibited a narrow pore-size distribution in the range of 0.5–3 nm (Fig. 1d, insert), indicating a substantial drug-loading capacity. X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical composition and valence states of CussNV. The survey spectra (Fig. 1e) revealed the characteristic peaks for Cu 2p (931.25 eV), O 1s (530.82 eV), N 1s (398.95 eV), C 1s (284.22 eV), and S 2p (161.34 eV). High-resolution XPS spectra of Cu 2p (Fig. 1f) showed peaks at 933.40 eV (Cu 2p₃_/₂) and 953.51 eV (Cu 2p₁_/₂), corresponding to Cu²⁺, while peaks at 931.30 eV (Cu 2p₃_/₂) and 951.25 eV (Cu 2p₁_/₂) indicated the presence of Cu⁺, with Cu⁺ comprising 62.24% and Cu²⁺ 37.76% of the total copper content (Fig. 1f, inset). The predominance of Cu⁺ was attributed to the reduction of Cu²⁺ during CussNV formation. As shown in Fig. 1g, high-resolution XPS spectra of S 2p displayed two main peaks at 161.18 eV (S 2p₃_/₂) and 167.58 eV (S 2p₁_/₂), which could be deconvoluted into three sulfur species, including S-S at 167.72 eV and 163.4 eV. Collectively, these results confirm the successful synthesis of CussNV.

Fig. 1

Characterizations of CussOMEp. (a) TEM image of CussNV. Scale bar is 20 nm. (b) Optimized atomic structure of the CussNV. Brow, red, blue, yellow, and the white balls denote Cu, O, C, S, and H atoms, repectively. (c) Electron density isosurface of the coordinated system. (d) N₂ adsorption-desorption isotherms and pore size distribution of CussNV. (e) XPS survey spectrum of CussNV. (f) High-resolution Cu 2p XPS spectrum of CussNV, with an inset showing the percentage of Cu²⁺ and Cu⁺ species. (g) High-resolution S 2p XPS spectrum of CussNV. (h, i) Hydrodynamic diameter (h) and zeta potential (i) measurements of CussNV, CussOME, and CussOMEp. (j) UV-vis absorption spectra of CussOMEp at different modification stages. (k) TEM images of CussNV after 10 h incubation with or without 10 mM GSH at pH 7.4 or 6.0. Scale bar is 40 nm. (l, m) Cumulative release profiles of copper (l) and OME (m) from CussOMEp dissolved in buffer containing 10 mM GSH at pH 7.4 and 6.0

We subsequently functionalized CussNV by loading OME and performing PEGylation through simple stirring. TEM images showed that both CussOME and CussOMEp retained a morphology similar to CussNV (Figure S2), indicating that the modifications with OME and DSPE-mPEG did not alter the structure. The atomic force microscopy (AFM) image indicated the nanosheet morphology of CussOMEp, with a thickness of less than 1.5 nm (Figure S3). The size from TEM images of CussNV, CussOME, and CussOMEp were 39.5 nm, 47.38 nm, and 52.51 nm, respectively (Figure S4). The hydrodynamic sizes of CussNV, CussOME, and CussOMEp were 41.8 nm, 50.4 nm, and 55.3 nm, respectively (Fig. 1h). As shown in Fig. 1i, the zeta potential shifted from − 5.22 mV for CussNV to -6.12 mV after OME loading. Following PEGylation, the zeta potential further decreased to -11.36 mV, attributable to the negatively charged -COOH groups in DSPE-mPEG. The UV-vis absorption spectra showed a characteristic peak at 278 nm for OME in CussOMEp (Fig. 1j), with a blue shift due to the chemical bond vibrations associated with OME binding to CussNV and the influence of solvent. Using the UV-vis standard curve of OME (Figure S5), the encapsulation efficiency was calculated to be approximately 18%. Fourier-transform infrared (FTIR) spectra (Figure S6) revealed characteristic peaks of DSPE-mPEG at 2916 cm⁻¹ (C-H) and 1106 cm⁻¹ (C-O) in CussOMEp. Additionally, all samples (CussNV, CussOME, and CussOMEp) displayed an S-S peak at 428 cm⁻¹, confirming the presence of disulfide bonds in CussNV. Peaks at 532 cm⁻¹ (S = O) were observed in both CussOME and CussOMEp, indicating the successful loading of OME.

We next investigated the biodegradability of CussNV in physiological and tumoral-mimicking conditions. As shown in Fig. 1k, in the pH = 7.4 PBS condition, CussNV exhibited minimal changes in morphology during 10 h incubation, while its morphology changed in the presence of 10 mM GSH. This indicates that CussNV degraded in the presence of GSH, which may arise from the thiol/disulfide exchange. In the acidic condition (pH = 6.0), CussNV underwent degradation, which was significantly accelerated in the presence of GSH. Complete collapse of the CussNV structure was observed under these conditions, highlighting its pH/GSH-responsive degradability and potential for tumor-specific therapeutic release. Thereafter, we further examined the release behaviors of OME and copper ions in CussOMEp. As can be seen from Fig. 1l and m, the release percentage of copper and OME from CussOMEp were both higher at pH 6.0 compared to pH 7.4, avoiding non-specificity leakage and achieving the pH-responsive tumor-specific therapy. The release percentages of copper (98.3%) and OME (98%) at pH 6.0 were substantially higher than the parallel of copper (67.2%) and OME (63.1%) at pH 7.4 in the presence of GSH.

The multienzyme mimetic properties of CussNV were thoroughly investigated in this study. First, we evaluated the GSHox-like activity of CussNV by incubating various concentrations of the nanovector with 10 mM GSH, using 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) as a specific probe to measure residual GSH levels (Fig. 2a). As shown in Fig. 2b, CussNV efficiently reduced GSH levels in a concentration-dependent manner, likely due to the oxidative action of Cu²⁺ and the thiol/disulfide exchange mechanism. This reaction followed typical Michaelis-Menten kinetics, with a calculated Michaelis-Menten constant (K_m) of 2.0 mM and a maximum initial velocity (V_max) of 2.34 × 10⁻⁶ mM s⁻¹ (Fig. 2c and S7).

Fig. 2

The enzymatic properties of CussNV. (a) Reaction of DTNB showing color changes before and after incubation with GSH. (b) UV-vis spectra of DTNB solutions and corresponding images after incubation with varying concentrations (0, 5, 10, 15, 20, 40, and 60 µg/mL) of CussNV for 4 h. (c) Michaelis–Menten kinetic analysis of the GSHox-like activity of CussNV. (d) Reaction of TMB showing color changes before and after incubation with H₂O₂. (e) UV-vis spectra of oxTMB incubated with 50 µg/mL CussNV, 0.5 mM TMB, and 100 µM H₂O₂ for 30 min and corresponding images of the oxTMB solutions. (f) Michaelis–Menten kinetic analysis of the POD-like activity of CussNV. (g) Catalytic decomposition of H₂O₂ by CussNV, resulting in the formation of water and oxygen. (h) Dissolved oxygen concentrations of CussNV incubated with 10 mM H₂O₂. (i, j) Equations (i) and mechanisms (j) for the GSHox-like, POD-like, and CAT-like activities of CussNV

Next, the POD-like activity of CussNV was assessed using a 3,3’,5,5’-tetramethylbenzidine (TMB) colorimetric assay, where ^•OH oxidize colorless TMB to its bluish oxidized form (oxTMB) (Fig. 2d). As demonstrated in Fig. 2e and S8, the absorbance of the TMB solution at 655 nm significantly increased in the presence of both CussNV and H₂O₂, compared to either CussNV or H₂O₂ alone, indicating that CussNV catalyzes the conversion of H₂O₂ to ^•OH. Additionally, CussNV degraded methylene blue (MB) and oxidized o-phenylenediamine (OPD) in the presence of H₂O₂ (Figure S9), with a K_m of 8.3 mM and a V_max of 3.59 × 10⁻⁶ mM s⁻¹ (Fig. 2f and S10), further confirming its strong POD-like activity. The electron spin resonance (ESR) spectra further evidenced the generation of ^•OH from CussNV utilizing the trapping agent DMPO (Figure S11). Furthermore, the CAT-like activity of CussNV was evaluated by measuring dissolved oxygen levels using an oxygen analyzer. As shown in Fig. 2g and h, after incubation with H₂O₂, CussNV produced a substantial amount of oxygen, demonstrating its CAT-like ability. These findings reveal that CussNV exhibits GSHox-, POD-, and CAT-like activities (Fig. 2i), enabling it to expand the ROS pool through its GSHox- and POD-like properties, while alleviating hypoxia through its CAT-like function (Fig. 2j). These multifunctional capabilities position CussNV as a promising candidate for enhancing ferroptosis, cuproptosis, and related antitumor immune responses.

Omeprazole inhibited copper excretion to enhance cuproptosis

We first investigated the cellular uptake behavior of CussNVp in the breast cancer 4T1 cell line. Fluorescein isothiocyanate-labeled CussNVp (CussNVp@FITC) was incubated with 4T1 cells for various durations, and intracellular fluorescence was analyzed using flow cytometry (FCM). As shown in Fig. 3a, green fluorescence intensity increased significantly with prolonged incubation, confirming the efficient uptake of CussNVp@FITC by 4T1 cells. Next, we assessed the cytotoxic effects of OME, and the results showed that OME induced moderate cell death in a concentration-dependent manner (Fig. 3b). The specific role of OME in ATP7A inhibition and Cu²⁺ excretion was visualized using confocal laser scanning microscopy (CLSM). Rhodamine B hydrazide (RBH) staining (Fig. 3c and S12) revealed that the OME group slightly increased cellular Cu²⁺ content without promoting ATP7A translocation, likely due to the low concentration of OME (10 µM) used in the experiment (Fig. 3d). In contrast, OME significantly increased Cu²⁺ levels in cells treated with CussNVp. CussNVp induced ATP7A dispersion and translocation from the Golgi to the extracellular space, likely due to elevated Cu²⁺ levels in 4T1 cells. However, CussOMEp did not cause ATP7A dispersion or translocation, resulting in enhanced Cu²⁺ accumulation. Quantitative analysis confirmed that CussOMEp-treated cells had significantly higher intracellular Cu²⁺ concentrations compared to other groups (Fig. 3e and S13).

Subsequently, we evaluated the generation of ^•OH induced by CussOMEp in 4T1 cells using the fluorescent probe BBoxiProbe O26. As shown in Fig. 3f and S14, cells treated with OME displayed slightly elevated green signals compared to the control (PBS) group. In contrast, CussNVp induced pronounced green signals, indicating a significant generation of ^•OH. Notably, CussOMEp produced even higher levels of ^•OH than CussNVp, correlating with increased Cu²⁺ levels. Additionally, CussOMEp led to a reduction in cellular GSH levels, as indicated by Bromodiamine staining (Fig. 3g and S15), likely due to its GSHox-like activity. The oxygen probe (Ru(ddp)₃Cl₂, RDPP) staining results (Fig. 3h and S16) revealed that both the control and OME groups exhibited strong green fluorescence, suggesting that OME had no significant impact on cellular oxygen content. Conversely, cells treated with CussNVp and CussOMEp showed dimmer green fluorescence, indicating that CussNVp enhances oxygen content through its CAT-like activity.

We further investigated the mechanisms of cell death in 4T1 cells induced by CussOMEp. Liperfluo staining (Fig. 3i and S17) demonstrated that CussOMEp- treated cells exhibited higher fluorescence signals compared to other groups, indicating the generation of lipid peroxides (LPO) and the occurrence of ferroptosis. Western blot analysis confirmed a significant reduction in GPX4 expression levels in CussOMEp-treated cells (Fig. 3j and S18), further supporting the occurrence of ferroptosis. Moreover, CussOMEp significantly increased the expression of heat shock protein 70 (HSP 70) and promoted oligomerization of dihydrolipoamide S-acetyltransferase (DLAT), while decreasing ferredoxin 1 (FDX1) expression, suggesting the induction of cuproptosis (Fig. 3k and S19).

Fig. 3

CussOMEp induces cell death in vitro. (a) Representative flow cytometric images of 4T1 cells treated with 50 µg/mL CussNVp@FITC for different time points (0, 1, 2, 4, and 12 h). (b) Viability of 4T1 cells exposed to varying concentrations (0, 1, 2, 5, 10, 20, 50, and 100 µM) of OME for 24 h. (c) Fluorescence images depicting intracellular Cu²⁺ levels in 4T1 cells following 10.8 µM OME, 60 µg/mL CussNVp, and 60 µg/mL CussOMEp for 24 h. scale bar: 10 μm. (d) Immunofluorescence images of ATP7A expression in 4T1 cells following 10.8 µM OME, 60 µg/mL CussNVp, and 60 µg/mL CussOMEp for 24 h, scale bar: 5 μm. (e) Quantification of intracellular Cu²⁺ concentrations (n = 3). (f) Fluorescent images of 4T1 cells stained with BBoxiProbe O26 (1:1000 dilution, 30 min, 37 ℃) to detect ^•OH. (g) CLSM images of GSH from 4T1 cells stained with bromobimane (100 µM, 30 min, 37 ℃). (h, i) Fluorescent images showing oxygen levels (h) and ferroptosis (i) in 4T1 cells stained with RDPP (50 µM, 2 h, 37 ℃)and Liperfluo (10 µg/mL, 30 min, 37 ℃), respectively, scale bar: 10 μm. (j, k) Western blot analysis of GPX4 (j), and HSP 70, DLAT, and FDX1 (k), in 4T1 cells under different treatments. (l) Cell viability of 4T1 cells incubated with various concentrations of CussNVp and CussOMEp (n = 5) for 24 h. (m, n) Flow cytometric analysis (m) and quantification (n) of apoptosis rates in 4T1 cells after different treatments. (o) Schematic illustration the mechanism of CussOMEp induces copper overload-mediated cuproptosis, ferroptosis, and apoptosis. Statistical significance denoted as *P < 0.5, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05), analyzed by one-way ANOVA, followed by Dunnett’s multiple comparisons test. Data represent mean ± s.d

We assessed the in vitro cytotoxicity of CussOMEp on 4T1 cell viability using a CCK-8 detection kit. As depicted in Fig. 3l, cells treated with CussOMEp exhibited reduced viability compared to the CussNVp group, indicating a synergistic effect of OME and CussNVp in promoting cell death. Furthermore, we examined CussOMEp-induced apoptosis using an Annexin V-FITC/PI detection kit (Fig. 3m and n). CussOMEp-treated cells exhibited an apoptosis rate of 29.3%, significantly higher than the rates observed in the OME (8.52%) and CussNVp (8.03%) groups. Collectively, these findings highlight that CussOMEp induces cell death in a synergistic manner through its multiple enzymatic properties (Fig. 3o), demonstrating significant potential for tumor therapy.

In vivo biodistribution

The CussNVp was labeled with the far-infrared fluorescent dye indocyanine green (ICG), thereafter referred to CussICGp, to visualize the in vivo biodistribution in 4T1 tumor-bearing mice. The loading efficiency of ICG in CussNV was determined to be 58.5% (Figure S20), with the equivalent ICG dosage used in two groups. The procedures are illustrated in Fig. 4a. As shown in Fig. 4b and c, the fluorescence signals from tumors in the free ICG group gradually decreased over time, nearly vanishing by 8 h post-injection. In contrast, the CussICGp group exhibited rapid accumulation in tumor regions, peaking at 4 h post-injection, indicating the exceptional tumor-targeting capacity of CussNVp. Ex vivo fluorescence images in Fig. 4d and e further confirmed the predominant tumor distribution of CussICGp compared to major organs (heart, liver, spleen, lung, and kidney).

Fig. 4

In vivo biodistribution of CussNVp (n = 3). (a) Schematic illustration of the treatment schedule. (b, c) Fluorescent images (b) and corresponding statistical analyses (a) of free ICG and CussICGp (100 µg ICG per mouse) in 4T1 tumor-bearing mice at different time points (n = 3). (d, e) Ex vivo fluorescence images (d) and statistical analysis (e) for major organs (He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney) and tumors (Tu) collected 48 h post-injection from mice treated with free ICG and CussICGp; yellow dashed circles indicate tumor locations (n = 3). (f) Cu²⁺ concentration at the tumor sites (n = 3). Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data are presented as mean ± s.d

In a separate experiment, we assessed Cu²⁺ levels in tumors following intravenous (i.v.) administration of PBS (control), OME, CussNVp, and CussOMEp. Notably, OME did not significantly elevate tumoral Cu²⁺ levels (Fig. 4f), likely due to its limited tumor-targeting ability. In contrast, tumors from mice injected with CussNVp exhibited significantly increased Cu²⁺ levels. Importantly, CussOMEp demonstrated the highest Cu²⁺ levels among the four groups, indicating its superior capacity for Cu²⁺ accumulation in tumors. These findings strongly support the remarkable tumor accumulation capability of CussNVp, underscoring its potential for achieving significant therapeutic effects.

In vivo biocompatibility of CussOMEp

Biocompatibility is essential for in vivo applications. Initially, we employed a hemolytic test to assess the safety of CussNVp and CussOMEp. As shown in Figure S21, the hemolysis rate did not exceed 5% across various concentrations of CussNVp and CussOMEp. To further evaluate the biosafety of CussOMEp, healthy female Balb/c mice were divided into two groups and administered intravenous injections of CussOMEp or an equal volume of PBS as a control (Figure S22a). Throughout the 90-day monitoring period, body weights increased steadily in both groups, with no significant differences observed (Figure S22b).

On day 90, the mice were sacrificed, and major organs were collected and weighed; serum samples were obtained for routine blood analysis. No significant differences in the visceral index of organs between the PBS and CussOMEp groups were noted, providing strong evidence for the promising biosafety of CussOMEp (Figure S22c). Additionally, routine blood examinations revealed no significant distinctions between the two groups (Figure S22d). Liver and kidneys are crucial for maintaining bodily functions, including the removal of metabolic byproducts and toxic waste, while retaining useful metabolites to sustain homeostasis. Moreover, the kidneys effectively cleared nanoparticles, mitigating toxicity associated with prolonged retention. Consequently, we measured hepatorenal function indices, including alanine aminotransferase (ALT), alkaline phosphatase (ALP), serum urea nitrogen (BUN), and serum creatinine (CR). As illustrated in Figures S22d, no significant differences were observed between mice treated with PBS and those treated with CussOMEp, confirming that CussOMEp nanoparticles did not impair hepatorenal function and exhibited remarkable biological safety. Hematoxylin and eosin (H&E) staining of principal organs further corroborated the biosafety of CussOMEp, showing no abnormalities in cell morphology (Figure S22e). In summary, these findings indicate the outstanding biocompatibility and biosafety of CussOMEp.

CussOMEp evokes antitumor immune responses

We established a murine model with 4T1 tumors to evaluate the synergistic therapeutic effects of CussOMEp. The treatment regimen is outlined in Fig. 5a. Mice were administered the designated formulations via i.v. injection on day 0, with body weight and tumor volume monitored every two days. On day 14, the mice were sacrificed for further analysis. As shown in Fig. 5b and c, treatment with OME alone had minimal impact on tumor growth compared to the control group (PBS). In contrast, CussNVp modestly delayed tumor progression. Notably, CussOMEp significantly inhibited tumor growth over the 14-day period. Tumor weights (Fig. 5d) and images (Fig. 5e) further confirmed the superior efficacy of CussOMEp, which achieved a tumor growth inhibition (TGI) rate of 71.6% after 14 days of treatment (Fig. 5f). Additionally, no significant changes in body weight were observed throughout the treatment period, indicating the excellent biocompatibility of the CussOMEp nanoplatform (Figure S23). Hematoxylin and eosin (H&E) staining of tumor sections (Fig. 5g and S24) revealed substantial cellular damage in the CussOMEp-treated group, including pyknotic nuclei, suggesting extensive cytotoxicity. Immunohistochemical (IHC) staining of tumor sections showed that CussNVp reduced the expression of hypoxia-inducible factor 1-alpha (HIF-1α) (Fig. 5g, h and S24), and this effect was further enhanced in the CussOMEp-treated group, indicating a more pronounced alleviation of hypoxic conditions. The superior hypoxia relief observed with CussOMEp may be attributed to the higher copper concentration in tumors, where copper ions catalyze the conversion of tumor H₂O₂ to O₂ during the catalytic reaction. Furthermore, tumors from CussOMEp-treated mice exhibited significantly lower expression of FDX1 and GPX4 (Fig. 5g, h and S24) compared to the CussNVp group and other control groups, suggesting that CussOMEp effectively induced both cuproptosis and ferroptosis in tumor cells.

Fig. 5

In vivo tumor therapy and immune response activation by CussOMEp (300 µg per mouse). (a) Schematic representation of the treatment schedule. (b, c) Tumor growth curves showing individual (b) and total (c) tumor sizes (n = 4). (d-f) Tumor weight (d), corresponding images (e), and TGI values (f) in mice post treatments on day 14. (g) H&E staining and IHC staining for HIF-1α, FDX1, and GPX4 in tumor sections. (h) Percentage of M2 macrophages identified by flow cytometry (gated on CD11b⁺F4/80⁺CD206⁺). (i, j) Flow cytometry plots (i) and statistical analysis (j) of DC maturation in lymph nodes (gated on CD11c⁺CD80⁺CD86⁺). (k-n) Quantitative results for CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), neutrophils (CD11b⁺F4/80⁺Ly6G⁺), and B cells (B220⁺). Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns (not significant, P > 0.05), determined by one-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean ± s.d

Lastly, we investigated the immune responses elicited by CussOMEp, focusing on macrophage polarization in tumors and dendritic cell maturation in lymph nodes. As shown in Figure S25, treatment with either OME or CussNVp reduced the percentage of M2 macrophages (CD11b⁺F4/80⁺CD206⁺) to 16.3% and 11.7%, respectively, compared to 21.1% in the control group. Notably, CussOMEp treatment resulted in a significant reduction of M2 macrophages to 9.8%, highlighting its strong effect on M2 macrophage polarization. In addition, the percentages of mature DCs (CD11c⁺CD80⁺CD86⁺) in the OME, CussNVp, and CussOMEp groups were 10.3%, 16.7%, and 21.8%, respectively (Fig. 5i and j, and S26). The highest level of DC maturation observed in the CussOMEp group suggests that CussOMEp effectively induces a robust immune response.

Since the maturation of DCs can regulate T cells proliferation, we next evaluated the percentages of helper T cells (CD3⁺CD4⁺) and cytotoxic T lymphocytes (CD3⁺CD8⁺) within tumors. As shown in Fig. 5k and l, and S27, mice treated with CussOMEp exhibited the highest proportion of tumor-infiltrating helper T cells and cytotoxic T lymphocytes. Additionally, CussOMEp treatment increased the percentage of neutrophils (CD11b⁺F4/80⁺Ly6G⁺) to 13.9% (Fig. 5m, S28, and S29) and B cells (B220⁺) to 29.2% (Fig. 5n, S30, and S31), surpassing the levels observed in other groups. These findings suggest that CussOMEp activates both adaptive and innate immune responses, highlighting its potential in tumor immunotherapy.

CussOMEp combined with immune checkpoint blockade to suppress metastasis

Recent advances in immune checkpoint blockade (ICB), particularly targeting the PD-L1/PD-1 axis, have revolutionized cancer therapy. However, the therapeutic efficacy of αPD-1 monotherapy is often constrained by the highly immunosuppressive tumor microenvironment (TME), which significantly diminishes its effectiveness. Achieving a durable and robust therapeutic response with minimal treatment frequency remains a formidable challenge in oncology. In this study. we demonstrated that that CussOMEp, a novel nanozyme system, effectively stimulates antitumor immune responses, providing a promising strategy to overcome these limitations. To further enhance its efficacy, we combined the CussOMEp with αPD-1 and investigated its potential to inhibit metastasis in a bilateral tumor model (Fig. 6a). As shown in Fig. 6b and c, primary tumors in the control group grew rapidly, while αPD-1 treatment delayed tumor progression. CussOMEp initially suppressed tumor growth but lacked long-term efficacy. In contrast, the combination of CussOMEp and αPD-1 resulted in significantly smaller primary tumors, highlighting a strong synergistic effect on tumor suppression.

Additionally, the CussOMEp + αPD-1 group exhibited robust inhibition of distant tumor growth, outperforming both the αPD-1 and CussOMEp monotherapy groups, which only exhibits moderate suppression. The inhibition ratios in the CussOMEp + αPD-1 group were 6/8, compared to 0/8 in the control group, 4/8 in the αPD-1 group, and 5/8 in the CussOMEp group (Fig. 6d and e). Throughout the treatment, minimal changes in mouse body weight were observed, indicating low toxicity (Figure S32). Notably, the combination treatment significantly extended survival. As shown in Figure S33, the survival rates after the treatment period were 37.5% in the control group, 75% in the αPD-1 group, 50% in the CussOMEp group, and 100% in the CussOMEp + αPD-1 group, demonstrating the superior efficacy of the combination therapy.

Fig. 6

In vivo therapeutic efficacy of CussOMEp (300 µg per mouse) combined with αPD-1 (1 mg/kg) in an abscopal tumor model. (a) Schematic representation of the treatment schedule. (b, c) Growth curves of primary tumors, presented as total (b) and individual sizes (c), n = 8. (d, e) Growth curves of distant tumors, shown as total (d) and individual sizes (e). (f-i) Percentages of DC maturation (f), M2 macrophages (g), M1 macrophages (h), and Tregs (i). (j-m) Statistical analysis of immune cell populations, including CD4⁺ T cells (j), CD8⁺ T cells (k), CD8⁺IFN-γ⁺ T cells (l), and effector T cells (m). Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Data are presented as mean ± s.d

We further investigated the immune response elicited by the CussOMEp + αPD-1 treatment in inhibiting abscopal tumors. The CussOMEp + αPD-1 group significantly enhanced DC maturation, increasing from 7.4 to 26.1% (Fig. 6f, S34, and S35). Additionally, the treatment induced a phenotypic shift in macrophages from the immunosuppressive M2 type to the immunostimulatory M1 type (Fig. 6g and h, and S36-S37). Specifically, the proportion of M2 macrophages at distant tumor sites decreased to 6.4% in the CussOMEp + αPD-1 group, compared to 18.7% in the control, 12.9% in the αPD-1 group, and 10.4% in the CussOMEp group. In contrast, the proportion of M1 macrophages increased to 23.5%, exceeding levels in the control group (9.0%), the αPD-1 group (17.7%), and the CussOMEp group (21.5%). These results indicate that CussOMEp + αPD-1 robustly activates the innate immune response.

T regulatory (Treg) cells, which exhibit immunosuppressive functions crucial for maintaining self-tolerance, were also analyzed (Fig. 6i, S38, and S39). Mice treated with CussOMEp + αPD-1 displayed a marked reduction in the ratio of Treg cells to 7.6%, compared to 18.0% in both the control and αPD-1 groups, and 12.0% in the CussOMEp group. Moreover, the helper T cells in the CussOMEp + αPD-1 group reached 10.5%, significantly higher than in the control (3.6%), αPD-1 (6.8%), and CussOMEp (8.6%) groups (Fig. 6j and S38). A similar trend was observed for cytotoxic T lymphocytes (Fig. 6k and S38). The percentage of CD8⁺ IFN-γ⁺ cells was 23.9% in the CussOMEp + αPD-1 group, compared to 6.5% in the control group, 11.7% in the αPD-1 group, and 14.6% in the CussOMEp group (Fig. 6l, S38), suggesting that the combination treatment promotes the activation of T cells in distant tumors.

Additionally, we observed a significant increase in the proportion of effector memory T cells in the spleens of mice treated with CussOMEp + αPD-1 (Fig. 6m, S40, and S41), which is associated with long-term tumor suppression. Collectively, these findings demonstrate that CussOMEp + αPD-1 induces potent immune responses, thereby effectively suppressing the emergence of distant tumors.

The therapeutic efficacy of the CussOMEp and αPD-1 combination was further evaluated in a mouse model of lung metastasis, with the treatment schedule depicted in Fig. 7a. As shown in Fig. 7b and c, CussOMEp combined with αPD-1 significantly inhibited primary tumor growth compared to the other three groups. To monitor metastasis, bioluminescence images were captured using an IVIS imaging system. In the control, αPD-1, and CussOMEp groups, the fluorescent signal progressively increased over time (Fig. 7d and e), indicating a growing number of metastatic nodules. In contrast, almost no fluorescence signal was detected in the CussOMEp + αPD-1 group, suggesting a strong suppression of metastasis. India ink staining (Fig. 7f) and H&E staining (Fig. 7g and S42) further confirmed a marked reduction in metastatic foci in lung tissues following treatment with the CussOMEp + αPD-1 combination. These results collectively demonstrate the superior anti-metastatic effect of CussOMEp in combination with αPD-1. Additionally, no significant body weight loss was observed (Figure S43), indicating the safety of the treatment. The synergistic CussOMEp + αPD-1 therapy also significantly extended long-term survival rates (Fig. 7h), further underscoring the efficacy of this combination therapy.

Fig. 7

Inhibition of lung metastasis by CussOMEp (300 µg per mouse) in combination with αPD-1 (1 mg/kg). (a) Schematic illustration of treatment schedule. (b, c) Growth curves showing total tumor burden (b) and individual tumor sizes (c) for all experimental groups (n = 8). (d, e) Representative bioluminescence images (d) and quantitative analysis (e) on days 21, 25, and 30. (f, g) Representative lung tissue images stained with India ink (f) and H&E (g), with tumors indicated (Tu). (h) Survival rates of mice under different treatment conditions. Statistical significance is indicated as **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns (not significant; P > 0.05), determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data are presented as mean ± s.d

Conclusion

In this study, we have successfully developed a functional nanozyme system, CussOMEp, which enhances tumor immunotherapy through the induction of both cuproptosis and ferroptosis. By integrating a copper-based nanovector (CussNV) with OME, a copper transporter inhibitor, this system not only promotes oxidative stress and cytotoxicity within tumor cells but also modulates the tumor microenvironment by alleviating hypoxia and depleting GSH. The multifunctional capabilities of CussOMEp, including POD, GSHox, and CAT-like activities, enable effective tumor killing through the generation of lethal hydroxyl radicals and the inhibition of copper transporter ATP7A. This results in enhanced accumulation of copper ions and the potentiation of ferroptosis and cuproptosis, leading to robust immunogenic responses. When combined with αPD-1 therapy, CussOMEp exhibits significant antitumor effects, reducing tumor metastasis and promoting immune cell activation, including DC maturation and T cell proliferation. Our results demonstrate that this nanozyme system is a promising strategy for overcoming the limitations imposed by dysregulated copper metabolism and hypoxic tumor environments, paving the way for improved outcomes in synergistic tumor immunotherapy. This work underscores the potential of leveraging metal-ion-induced cell death pathways as part of an integrated approach to cancer treatment, offering a transformative platform for future therapeutic developments.