Biomimetic nanoplatform with multienzyme cascade activity boosting ROS generation and immune activation feedback for tumor therapy

TL;DR

A new biomimetic nanoplatform boosts tumor treatment by combining ROS generation and immune activation through a cascade of enzyme-like activities.

Contribution

The first reported glutathione oxidase- and L-cysteine oxidase-like activities of nano-realgar in tumor therapy.

Findings

The nanoplatform's cascade catalytic effect enhances tumor suppression through ROS generation.

ROS and immune activation create a positive feedback loop improving treatment outcomes.

Abstract

Nanocatalytic medicine has emerged as a promising strategy for tumor therapy, utilizing nanozymes to generate cytotoxic reactive oxygen species (ROS). Despite significant progress, challenges persist, including low catalytic efficiency and inadequate tumor targeting. Herein, we report a tumor cell membrane coated biomimetic nanozyme platform (CMNP) containing manganese dioxide nanoparticles (MnO2@BSA), nano-realgar (NR), and doxorubicin (DOX). This nanoplatform targets tumors via biomimetic properties. In the tumor, MnO2 alleviates hypoxia by catalytically decomposing H2O2 to produce O2, thereby enhancing the enzymatic activity of NR. As the nanocrystalline form of a traditional Chinese medicine, NR exhibits glutathione oxidase (GSHOx)- and L-cysteine oxidase (LCO)-like activities, generating substantial cytotoxic ROS. These cascade catalysis-enhanced ROS not only induce tumor cell apoptosis but also activate immune responses in the tumor microenvironment. Furthermore, released DOX and Mn2+ ions recruit tumor-killing immune cells by inducing the immunogenic cell death (ICD) of tumor cells and activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, respectively. The tumor suppression stems from a positive feedback regulation driven by ROS generation and immune activation. Our study proposes a versatile nanoplatform that offers a two-pronged treatment strategy.

We report a biomimetic nanozyme platform (CMNP) containing manganese dioxide nanoparticles (MnO2@BSA), nano-realgar (NR), and doxorubicin (DOX) coated with tumor cell membrane. The tumor suppressive effects with CMNP treatment are amplified via synergism between the nanocatalytic cascade effect and the two-pronged treatment strategy. CMNP offers a novel and valuable approach for treating tumor.Image 1

•The Glutathione oxidase- and L-cysteine oxidase-like activities of nano realgar are first reported.•The cascade catalytic effect of multiple nanoenzymes promotes tumor treatment.•The positive feedback regulation of ‘oxidative stress-ferroptosis-immune activation’.

The Glutathione oxidase- and L-cysteine oxidase-like activities of nano realgar are first reported.

The cascade catalytic effect of multiple nanoenzymes promotes tumor treatment.

The positive feedback regulation of ‘oxidative stress-ferroptosis-immune activation’.

Figures

a, b, 7c, 7d, 7e, 7](#fig7)f and S31). The flow cytometry analysis of T cells in the spleen showed that, compared with the saline group, the CD8^+^ T and CD4^+^ T cells increased by 176% and 135% respectively after CMNP treatment. Similar changes were also observed in the peripheral blood. After CMNP treatment, the number of CD8^+^ T cell and CD4^+^ T cells increased by 149% and 148% respectively, indicating that CMNP can induce the activation of T cells. Especially the proportion of CD4^+^ T cells was 40.8%, which was significantly higher than the rate in the PBS group in tumor (23.6%) (Fig.

Full text

Introduction

1

Nanocatalytic medicine has rapidly developed into an effective strategy for cancer therapy [1]. Unlike conventional chemotherapy, which targets rapidly dividing cells, nanocatalytic approaches induce apoptosis in tumor cells via elevated oxidative stress generated by nanocatalysts [2]. However, this therapeutic approach has some limitations, such as low catalytic activity and therapeutic efficacy [3]. Thus, there is an urgent need to identify nanocatalysts with desirable therapeutic efficacy. Nanozymes are commonly used as nanocatalysts because of their multi-enzyme-like activity and excellent stability in biological microenvironments [4,5]. These nanozymes also exhibit the characteristics of nanomaterials, such as the enhanced permeability and retention (EPR) effect [6], which effectively improves the ability to target tumors [3]. A cascade effect was made possible by the synergistic catalysis of nanozymes due to their multi-enzyme-like activities. Tumor cells were directly destroyed by this enzymatic cascade, which increased oxidative stress and produced significant amounts of reactive oxygen species (ROS) in the tumor microenvironment [7].

Since Fe_3_O_4_ nanoparticles were defined as nanozymes in 2007 by Yan's group [8], multiple types of nanozymes with metallic and nonmetallic elements have been reported and used in tumor therapy. The catalytic activity of nanozymes can be regulated by adjusting particle size or morphology, which can in turn enhance ROS production. In general, smaller particle sizes are associated with higher catalytic activity [9]. Alternatively, appropriate surface modification can improve the ability of nanozymes to perform active targeting [10]. Moreover, nanozyme-based nanocatalytic therapy can be combined with other therapeutic strategies such as immunotherapy to synergistically improve the treatment efficacy [11]. Some transition metal oxide nanozymes can not only produce ROS to kill tumor cells but also release metal ions to activate the immune response [12]. For example, it has been reported that MnO_2_ metal nanozyme can catalyze H_2_O_2_ decomposition to produce O_2_ in the tumor [13,14]. Meanwhile, the released Mn^2+^ ions can activate the cyclic guanosine monophosphate adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway of dendritic cells (DCs) during antitumor immunotherapy [15]. Unlike immunotherapy strategies driven by sonodynamic and chemodynamic therapy [[16], [17], [18]], the cGAS-STING pathway directly recognizes DNA and triggers a powerful adaptive T-cell immune response, and is expected to form immune memory. In contrast, sonodynamic therapy and chemodynamic therapy mainly activate the immune response indirectly and usually to a relatively limited extent by inducing tumor cell immunogenic death [19,20]. Therefore, the advantage of the cGAS-STING pathway lies in being an efficient ‘starter’ of immunity. It provides a unique and powerful strategic foundation to establish a persistent and systematic antitumor immunity.

Besides oxide nanoparticles, sulfide nanoparticles also exhibit activities that resemble those of enzymes [21]. For example, the nanocrystallization of realgar, an arsenic sulfide compound used in traditional Chinese medicine, can improve its biocompatibility for biomedical uses [22]. It was shown that realgar nanoparticles can be used to treat leukemia by inducing apoptosis [23,24]. However, to the best of our knowledge, no study has reported the nanozyme activity of realgar nanoparticles. The arsenic valence state of realgar nanoparticles can be transformed from As^2+^ to As^3+^ with the vacancy effect. The enzyme-like activities of realgar nanoparticles are attributed to these changes in valence accompanied by electron transfer. Additionally, sulfides are highly chemically active, raising the possibility that realgar nanoparticles can exert a variety of catalytic effects. In the tumor microenvironment, the overexpression of glutathione (GSH) promotes tumor proliferation and metastasis, while GSH depletion can slow tumor growth [25]. Because of the reducibility of GSH, realgar nanoparticles can potentially act as glutathione-like oxidase (GSHOx) to decrease GSH content [26].

To study the therapeutic efficacy of nanodrugs for breast cancer, 4T1 cells were selected to model tumor growth and development in mice [27]. Breast cancer is a malignant tumor with a high mortality rate that is commonly treated by chemotherapy [28], particularly using doxorubicin (DOX) [29]. However, DOX is limited by its low targeting ability and severe side effects. To improve its therapeutic efficacy, DOX can be delivered to tumor sites by nanocarriers. Moreover, compared with the passive targeting of nanocarriers, an ability to target DOX to the tumor cell membrane actively can prolong its time in circulation. This is also valuable as tumor cell membrane modification in this way can help to avoid the clearance of nanoparticles by the immune system. The accumulation of DOX in the tumor can trigger immunogenic cell death (ICD) and stimulate the release of damage-associated molecular patterns (DAMPs), amplifying the effect of immune therapy [30,31].

In view of this background, we engineered a tumor cell membrane-coated bionic nanozyme platform (CMNP) for use in tumor therapy (Scheme 1). Bovine serum albumin (BSA) modified MnO_2_ nanoparticles (MnO_2_@BSA) and ethanolamine-modified nano-realgar (NR) served as the nanozyme on which DOX was added, forming a drug-loaded nanocomplex (MnO_2_@BSA/NR/DOX, MNP). MNP was then coated with 4T1 cell membranes, forming CMNP. The biomimicry of CMNP confers tumor-targeting ability. MnO_2_@BSA can react with endogenous H_2_O_2_ in the tumor microenvironment to produce O_2_, which alleviates tumor hypoxia and activates glutathione oxidase (GSHOx)- and L-cysteine oxidase (LCO)-like activities of NR. The cytotoxic ROS generated by a cascade of nanozyme activities could induce tumor cell apoptosis and activate immune responses. In addition, the CMNP-induced activation of ICD in tumor cells and the cGAS-STING signaling pathway can promote the accumulation of immune cells in the tumor microenvironment, specifically involving the phenotypic switch of macrophages to the M1 subtype and elevated proportions of CD8^+^ and CD4^+^ T cells. Overall, the synergistic effects of nanocatalytic medicine and the two-pronged treatment strategy can effectively suppress tumor growth.Scheme 1. Schematic illustration of the synthesis route of CMNP and the mechanism of multi-enzyme activity combined with immunotherapy in vivo.Scheme 1

Experimental section/methods

2

Materials

2.1

All the used reagents are purchased from agents and have not undergone any pre-treatment. Bovine serum albumin (BSA) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (China). Potassium permanganate (KMnO_4_), Hydrochloric acid (HCl), Nitric acid (HNO_3_), Hydrogen peroxide (H_2_O_2_), acetic acid, NaOH, Disodium hydrogen phosphate dodecahydrate, Sodium dihydrogen phosphate dihydrate, and Sodium sulfite were purchased from Sinopharm Chemical Reagent Co., Ltd (China). Sodium acetate trihydrate was purchased from Shanghai Haohong Scientific Co., Ltd (China). Doxorubicin hydroch (DOX) was purchased from Shanghai Macklin Biochemical Co.,Ltd (China). 3,3′,5,5′-tetramethylbenzidine (TMB), Sigma Aldrich Trading Co., Ltd (Germany). Trypsin-EDTA (0.25%), BCA kit, DAPI staining solution, Calcein AM/PI Cell Apoptosis Kit, and Annexin V/PI Cell Apoptosis Kit were brought from Shanghai Beyotime Biotechnology Co., Ltd (China). NADH Oxidase (NOX) Activity Assay Kit, Reduced Glutathione (GSH) Content Assay Kit were purchased from Beijing Solarbio Science&Technology Co., Ltd (China). L-Cysteine was purchased from Shanghai Adamas Reagent Co., Ltd (China). Hydrogen Peroxide Assay Kit was purchased from Nanjing Jiancheng Bioengineering Research Institute (China). Sulfo-Cyanine5.5 NHS ester was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (China). Annexin V-FITC/PI Cell Apoptosis Kit were brought from Thermo Fisher Scientific (USA). Fetal bovine serum (FBS) were purchased from Gibco (USA). Cell Counting Kit-8 (CCK-8), DMEM high glucose medium were brought form Melone Pharmaceutical Co., Ltd (China). Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F12), Roswell Park Memorial Institute medium 1640 (RPMI-1640) were purchased from Jiangsu Keygen Biotech Co., Ltd (China). Collagenase was purchased from Absin Bioscience Inc (China). Anti-glutathione peroxidase (GPX4) was purchased from Abmart (China). anti-STING/anti-p-STING, and anti-IRF3/anti-p-IRF3 antibody were purchased from Signalway Antibody LLC (USA). HMGB1 and CRT were purchased from Biodragon (China). β-Actin was purchased from Proteintech Group, Inc (China). GAPDH was purchased from Bioworld Technology Co., Ltd. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 568) was purchased from Abcam plc (UK). APC-anti-CD3e, and PE-anti-CD8a, PerCP-Cy^TM^5.5-anti-CD11b were purchased from Becton, Dickinson and Company (USA). FITC-anti-CD4, APC/Cyanine7-anti-CD45, FITC-anti-F4/80, PE-anti-CD86, and APC-anti-CD206 were purchased from Biolegend (USA). Enzyme-linked immunosorbent assay (ELISA) were purchased from Jiangsu Meibiao Biotechnology Co., Ltd (China). The 4T1 cells and RAW 264.7 cells used during the experiments were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Synthesis of CMNP

2.2

The CMNP nanoparticles were obtained by fusing 4T1 cell membranes with the nanoparticles (MNP) by physical extrusion method (200 nm polycarbonate membrane). Before concentration, the DOX, NR, and MnO_2_@BSA without coupling on CMNP were separated via ultrafiltration (30 and 100 kDa). The concentrations of metal ions and DOX were detected by ICP-MS and UV respectively. The content of As and Mn in the nanoparticles (including MnO_2_@BSA/NR, MNP and CMNP) were 0.1 mg/mL and 1.7 mg/mL, respectively. The content of DOX was 13.6 μg/mL in CMNP and MNP. The concentration of DOX in DOX treated groups both in vivo and in vitro were in accordance with the MNP and CMNP treatment groups.

Enzyme-like activity of CMNP

2.3

The OXD-like enzymatic activity assays of CMNP were measured at room temperature using TMB (0.5 mM) in 0.02 M buffer solutions.

Using a dissolved oxygen electrode (JPSJ-605F, Shanghai Yi Electrical Scientific Instruments Co., LTD), the produced O_2_ was measured to evaluate the CAT-like enzymatic activity of CMNP. In 0.02 M buffer solutions, H_2_O_2_ (100 μM) was added in addition to various nanoparticles and concentration of manganese for measurement.

Apoptosis assay for CMNP

2.4

A 6-well plate was seeded with 2 × 10^5^ logarithmic growth phase 4T1 cells. The experiments were divided into different treatment groups. After 24 h incubation, the cells were collected and stained by Annexin V and PI. The apoptosis was analyzed by flow cytometry (Celesta, BD, USA). Additionally, the cells were collected and stained by Calcein AM/PI. The apoptosis was analyized by fluorescent microscope.

Animal model

2.5

The animal experiments carried in this research were approved by Ethics Committee of Xuzhou Medical University with Experimental Animal Regulations of Xuzhou Medical University (IACUC Number: 202209S27). The mice (BALB/c, female, 5-7 weeks) were supplied from the Experimental Animal Centre of Xuzhou Medical University. The 4T1 cells (5 × 10^6^) were subcutaneously injected into the back of the mice. After 5 days later, the size of tumor tissue grew to 50 mm^3^.

The treatment protocol of animals in vivo

2.6

The 4T1 tumor-bearing mice were divided into 5 groups (n = 5) randomly and treated with PBS (Control), DOX, MnO_2_@BSA/NR, MNP and CMNP. The drugs were intravenously injected into mice every 3 days. The volume size of tumor and the weight of mice were recorded every day.

Haemolytic test of CMNP

2.7

Rats blood was taken from the rats. Various concentration of CMNP were incubated with a 10% red blood cell suspension for 24 h. The absorption value was obtained at 540 nm. The saline group was utilized as a negative control. The distilled water group was utilized as a positive control.

The antitumor immune response in vivo

2.8

The tumor tissues were homogenized with PBS to generate the single-cell suspensions. For detection of T cells in tumors, the cell suspensions were stained with APC/Cyanine7-anti-CD45, FITC-anti-CD4, APC-anti-CD3e, and PE-anti-CD8a, and then quantitatively analyzed using flow cytometry. Additionally, for detection of M1 and M2 macrophages in tumors, the single-cell suspension of tumors were stained with APC/Cyanine7-anti-CD45, PerCP-Cy^TM^5.5-anti-CD11b, FITC-anti-F4/80, PE-anti-CD86, and APC-anti-CD206, and then quantitatively analyzed using flow cytometry (Celesta, BD, USA). The analysis of cytokines, including IL-2, TNF-α and IFN-γ, was measured with enzyme-linked immunosorbent assay (ELISA).

Statistical analysis

2.9

All the quantitative data were expressed as mean ± s.d. The differences between the two groups were performed by the Student's t-test. The significances among groups were detected by one-way analysis of variance (ANOVA). GraphPad Prism, FlowJo, and ImageJ were used for statistical analysis and figure production. The statistical significance was indicated as P > 0.05 (no significance, ns), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Results and discussion

3

Synthetic characterization of the CMNP

3.1

The synthesis of CMNP is illustrated in Fig. 1a. MnO_2_@BSA and NR served as nanozymes, which suppressed tumor growth via the production of ROS. BSA acts as a biological template to mineralize KMnO_4_ into MnO_2_ nanoparticles. Meanwhile, the presence of large numbers of carboxyl groups on BSA was exploited to provide a site where MnO_2_ and ethanolamine-modified NR could be connected. The conventional chemotherapeutic drug (DOX) was also added to the nanozyme system via van der Waals forces to form a drug-loaded nanocomplex (MNP). The MNP was then coated with 4T1 cell membranes to form CMNP; this CMNP could target and accumulate at the tumor site via its biomimicry. The successful synthesis of CMNP was determined by transmission electron microscopy (TEM) and a dynamic light scattering system. TEM revealed a uniform spherical structure of the synthesized nanoparticles. The diameter of both MnO_2_ and NR were less than 5 nm (Fig. S1) After MNP synthesis, the size increased to 50 nm (Fig. S2), while the final product CMNP (either negatively stained or not) were about 100 nm (Fig. 1b and Fig. S3). This increased size change was in agreement with the rise of hydrodynamic size between MnO_2_ and CMNP. As shown in Fig. 1c, the hydrodynamic size of CMNP was 136.03 ± 14.8 nm, which was much larger than that of MnO_2_@BSA (19.43 ± 0.5 nm). This different result in size was attributed to the measuring method. In TEM measurement, the size was tested by the core of nanoparticles. However, the size of nanoparticles by DLS was tested with hydration shell and the core of nanoparticles. Because the molecular weight of BSA was 66 kDa, the thicker hydration shell exists on the surface of nanoparticles which leads to the large size tested in DLS. Furthermore, the successful synthesis of MnO_2_ and NR was confirmed by the X-ray powder diffraction (XRD) pattern (Fig. S4), since the characteristic diffraction peaks of MnO_2_ and NR were observed. As MnO_2_, NR, and DOX were joined by electrostatic interaction, zeta potential analysis (Fig. 1d and Table S1) was performed to reveal the change in surface charge during the synthetic procedure. The results revealed an increase in zeta potential from −18.9 ± 0.8 mV (MnO_2_@BSA) to −8.2 ± 0.4 mV (MNP) due to the positively charged NR and DOX. The zeta potential of CMNP decreased to −15.3 ± 0.1 mV, which confirmed successful cell membrane coating and nanoparticle stability. We also tested Fourier transform infrared (FTIR) spectrum of MnO_2_@BSA (Fig. S5). The combination of metal and oxygen could be detected at 419 cm-1, which indicated that the MnO_2_ interacted with BSA. Because NR and DOX interact with BSA through electrostatic adsorption, no new peaks were detected in the infrared spectrum of MnO_2_@BSA/NR. TEM mapping also revealed the presence of different elements in CMNP (Fig. 1e and Fig. S6). Particularly, C, O, Mn, and As atoms were found to overlap inside the same nanoparticles. The UV-Vis absorption spectra of the different nanoparticles were determined (Figure S7, S8 and S9). These results also confirmed the successful synthesis of CMNP because similar characteristic peaks appeared in different nanoparticles. Given the biomimetic status of this cell membrane in the nano-system, its function should be tested. CD44 is a receptor protein on the surface of the tumor cell membrane, which plays important roles in the division, proliferation, and migration of cancer cells [32]. In order to clarify the cell membrane modification, we detected the expression of CD44 in 4T1 cells, MNP, and CMNP using western blot experiments (Fig. S10). After cell membrane coating, significant CD44 protein bands appeared in 4T1 cells and CMNP. Finally, we conducted X-ray photoelectron spectroscopy (XPS) analysis to characterize the valence state of elements in CMNP (Fig. 1f and S11). XPS analysis indicated that Mn existed in a mixture of valence states of Mn^4+^ (653.8 eV) and Mn^2+^ (641.5 eV), while As ion maintained the As^2+^ and As^3+^ forms required for therapeutic activity (Fig. 1g and h). This confirmed our hypothesis that the enzyme-like activities of the realgar nanoparticles are attributable to these changes in valence accompanied by electron transfer. In addition, CMNP exhibited excellent dispersion and stability in water, PBS and serum(Fig. S12), supporting its long circulation time in vivo. The released Mn^2+^, As^2+^, and DOX were tested at different pH conditions and the release curves are shown in Fig. 1i, j, and 1k. The contents of Mn^2+^, As^2+^ and DOX released from CMNP increased with time prolong. Compared with physiological environment (pH 7.4), the released amounts of drug and different ions were higher in tumor microenvironment (pH 6.5 and 5.5). Under acidic conditions, the CMNP exhibit enhanced release of higher drug payloads, which substantially enhanced its efficacy in eliminating tumor tissue. In addition, the drug release curves of CMNP were determined in 2 mM and 10 mM GSH conditions (Fig. S13). Compared with the lower condition of GSH, the drugs, including DOX, Mn, and As, were released faster in higher conditions. In the same pH condition, the drug release rate was slower in the GSH-free condition.Fig. 1Characterizations of CMNP. (a) Schematic diagram of the synthesis process of CMNP. (b) TEM images of CMNP. Inset: the diameter distribution of CMNP. (c) The hydrodynamic sizes of MnO_2_@BSA, MnO_2_@BSA/NR, MNP, and CMNP (n = 3). (d) Zeta potentials of MnO_2_@BSA, MnO_2_@BSA/NR, MNP, and CMNP (n = 3). (e) Elemental mapping analysis of CMNP. (f) XPS spectra of CMNP. (g) High-resolution spectra of Mn 2p in CMNP. (h) High-resolution spectra of As 3d in CMNP. Release curves of Mn, As and DOX from CMNP at (i) pH 5.5 buffer solution, (j) pH 6.5 buffer solution, (k) pH 7.4 buffer solution. (n = 3). Data are presented as mean ± s.d.Fig. 1

Functional characterization of the CMNP

3.2

After systematically characterizing the physicochemical properties of CMNP (e.g., particle size, potential, and stability), we further evaluated its catalytic activity to clarify whether it could act as a nanozyme that exerts the activities of multiple enzymes (Fig. 2a). The high level of endogenous hydrogen peroxide (H_2_O_2_) and the hypoxia in the tumor microenvironment promoted the proliferation and metabolism of tumor cells [33]. Therefore, consuming H_2_O_2_ and alleviating hypoxia in tumor by CMNP can effectively slow tumor growth. In our previous study [34], MnO_2_@BSA showed excellent catalase (CAT) activity, and MnO_2_@BSA was also an important component of CMNP in this study. Therefore, the concentration of dissolved O_2_ was determined in different pH conditions, as shown in Fig. 2b, c and 2d. The three conditions represent different physiological environments in vivo. O_2_ production could be detected with CMNP in different pH conditions, while more O_2_ was produced in an acidic environment. H^+^ may increase the electron-donating capacity, which speeds up the production of O_2_ in the acidic environment. Excellent oxidase (OXD) activity was also detected in CMNP (Fig. 2e, f and 2g). CMNP could catalyze tetramethylbenzidine (TMB) under different pH conditions, and this catalytic ability was positively correlated with CMNP concentration (Fig. S14). We also determined the H_2_O_2_ level consumed both exogenously and endogenously (upon incubation with 4T1 cells). As shown in Fig. 2h, the reduction of H_2_O_2_ could be tested when MnO_2_ was present in different materials and >50% H_2_O_2_ could be consumed with CMNP compared with the PBS-treated group. This enzymatic activity was also reflected in the tumor cells (Fig. S15).Fig. 2The functional verification of CMNP in vitro. (a) Schematic diagram of catalytic mechanisms of different types of nanoenzymes. The CAT activity of CMNP at (b) pH 5.5 buffer solution, (c) pH 6.5 buffer solution, (d) pH 7.4 buffer solution. The OXD activity of CMNP at (e) pH 5.5 buffer solution, (f) pH 6.5 buffer solution, (g) pH 7.4 buffer solution. (h) The image of H_2_O_2_ depletion by different materials (n = 3). (i) Electron paramagnetic resonance (EPR) spectra of CMNP. PBS was set as a blank. (j) The image of glutathione depletion by different materials (n = 3). (k) The image of cysteine depletion by different materials (n = 3). Data are presented as mean ± s.d. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001; compared with PBS group.Fig. 2

Besides the alteration the tumor microenvironments by CAT activity of CMNP, the generation of cytotoxic ROS by multi-enzyme like activities, such as the POD activity of CMNP could also inhibit the tumor growth. To verify the occurrence of POD activity, we detected the existence of hydroxyl radicals (·OH), the results of which are shown in Fig. 2i. The characteristic peaks were obtained in the electron spin resonance spectra when CMNP was treated with H_2_O_2_. This overproduced ·OH could directly induce cell death. Additionally, the glutathione (GSH) was also present at a high level and this overproduced GSH promotes tumor proliferation and inhibits ferroptosis in tumor cells. The decrease of GSH level could be achieved by GSHOx. To the best of our knowledge, this is the first report of NR exhibiting GSHOx-like activity and that the level of GSH declined upon treatment with NR-containing nanoparticles (Fig. 2j). The level of GSH could also be reduced indirectly by eliminating Cys, which is an amino acid that is essential for GSH synthesis. As shown in Fig. 2k, the level of Cys decreased after treatment with NR-containing nanoparticles. This indicated that the NR also exerted LCO-like activity. Meanwhile, the GSHOx-like and LCO-like activities of CMNP were stronger than those of NR. This can be explained by the fact that the O_2_ generated by MnO_2_@BSA could accelerate the catalytic ability of oxidase along with the cascade effect between MnO_2_@BSA and NR. To confirm the glutathione oxidase (GSHOx)- and L-cysteine oxidase (LCO)-like activities of different nanoparticles, we supplemented the relevant enzymatic kinetics studies (including MnO_2_@BSA, NR and CMNP) and the Michaelis constant (K_m_) and maximum reaction rate (V_max_) of different nanoparticles were also calculated (Figs. S16 and 17). Because the smaller value of K_m_ indicated the stronger substrate affinity, the NR exhibited the best binding capacity with GSH and L-Cys. These results demonstrated the nanozyme-like catalytic activity of NR. Additionally, the GSHOx- and LCO-like activities of MnO_2_@BSA were verified due to the values of K_m_ were 0.4132 mmol/L and 0.2748 mmol/L for GSH and L-Cys, respectively. Compared with MnO_2_@BSA and NR, CMNP exhibited weaker substrate affinity, which was attributed to the influence of its cell membrane-coated surface on binding activity. However, CMNP exhibited the highest V_max_ in both GSHOx- and LCO-like activities, demonstrating that component synergy is the dominant contributor to catalytic efficiency enhancement in cascade catalysis.

Therapeutic effect of CMNP in vitro

3.3

The cellular uptake of MNP and CMNP was investigated in 4T1 cells with confocal laser scanning microscopy (Fig. 3a and S18). Cy5.5-labeled MNP and CMNP were prepared by labeling with the red fluorophore Cy5.5, and notably, the intracellular fluorescence signal intensities were significantly enhanced after 8 h of incubation. Compared to MNP, cells exhibited more rapid uptake of CMNP; meanwhile, stronger red fluorescence signals at the 8-h time point reflected a higher intracellular accumulation of CMNP. The quantification of cellular uptake of CMNP was confirmed with flow cytometry (Figs. S19 and S20). The nanoplatform was phagocytized by cells with time-depended effect. The same result was observed in the Bio-TEM images. After 8 h of incubation of 4T1 cells with CMNP (Fig. 3b), CMNP could be detected in both death and alive cells. This proved that CMNP could enter the cell through endocytosis and exert its function within the cell. As shown in Fig. 3c and d, CMNP exhibited dose-dependent cytotoxicity. Lower cell viability was shown in the CMNP-treated group than in the other treatment groups. This indicated that the internal components of CMNP could synergistically reduce the viability of cells. This decreased cell viability was due to apoptosis, which was detected by flow cytometry in the different treatment groups (Fig. 3e). The findings revealed clear apoptosis in the CMNP-treated group, with this effect also being positively correlated with CMNP concentration. Imaging of the fluorescence staining also revealed similar results (Fig. S21), indicating that CMNP could effectively induce apoptosis. The cytotoxicity of CMNP in RAW 264.7 was detected in Fig. S22. The cell viability treated with the nanoplatform was similar to PBS-treated groups, which indicated the biocompatibility of CMNP. We elucidated the potential mechanism of cell deaths promoted by CMNP (Fig. 3f). The over produced ROS in cells was the main factor that leading tumor cell death. Intracellular ROS levels of 4T1 cells were examined in the different treatment groups by fluorescence microscopy and flow cytometry (Fig. 3g–S23 and S24). The ROS level of CMNP-treated cells was higher than those of the other treatment groups.Fig. 3The cytotoxicity and analysis in vitro. (a) Confocal laser scanning microscope (CLSM) images. (b) Bio-TEM images of death and alive 4T1 tumor cells after CMNP treatment. Cytotoxicity analysis of 4T1 cells receiving (c) different materials and (d) concentration for 24 h (n = 5). (e) Annexin V-FITC/PI staining of 4T1 cells receiving different materials and concentration using flow cytometry. (f) Schematic diagram of potential mechanism of tumor cell death. (g) Quantification of ROS level in different treatment groups. (h) Western blotting analysis of GPX4 expression in 4T1 cells after different treatments. (i) Quantitative analysis of GPX4 (n = 3). (j) The image of NADH depletion by different materials (n = 3). Data are presented as mean ± s.d. P > 0.05 (no significance, ns), ∗∗∗∗P < 0.0001; compared with PBS group.Fig. 3

This overproduction of ROS was also attributed to ferroptosis induced by CMNP. To confirm this, ferroptosis was determined by measuring the glutathione peroxidase 4 (GPX4) level in the different groups (Fig. 3h and i). GPX4 protein expression was found to decrease in the CMNP-treated group, indicating that CMNP could consume intracellular GSH and lead to cell ferroptosis. This enhanced ferroptosis was also confirmed by testing intracellular ROS levels, given that ferroptosis could be associated with ROS production. Meanwhile, an increase in ROS level was related to the reduction of intracellular NADH. NADH is an important antioxidant, and the reduction of intracellular NADH inhibits cell proliferation, differentiation, and metabolism [35]. The results revealed a lower level of NADH in the cells treated with CMNP, which also indicated the inhibition of tumor cell growth by CMNP (Fig. 3j). The principle of nanocatalytic medicine is to utilize the abnormal oxidative stress microenvironment of tumors (high levels of H_2_O_2_) to trigger catalytic reactions (such as Fenton/Fenton-like reactions) and generate ROS, thereby achieving selective killing of tumor cells [1]. In our research, the ROS generation by Mn^2+^- and As^3+^-based nanozyme cascade catalysis was one of the therapeutic mechanisms. MnO_2_ alleviated hypoxia by catalytically decomposing H_2_O_2_ to produce O_2_, thereby enhancing the enzymatic activity of NR. As the nanocrystalline form of a traditional Chinese medicine, NR exhibited glutathione oxidase (GSHOx)- and L-Cysteine oxidase (LCO)-like activities, generating substantial cytotoxic ROS. While previous research has documented As^3+^’s direct anti-tumor effect [22], the nanozyme activities of As^3+^ in CMNP were the main mechanism in this research. In particular, As^3+^ in CMNP could function as a nanozyme to initiate cascade catalytic reactions in the tumor microenvironment, which could effectively and continuously produce ROS (more potent than the direct killing effect of As^3+^ alone). The fundamental mechanism of the As^3+^-mediated anti-tumor effect in our investigation was this cascade catalytic process based on the nanozyme effect.

The accumulated ROS could also induce the immunogenic cell death (ICD) of tumor cells which synergetic enhanced the immune response with DOX. In order to determine the ICD in tumor cells, we detected the expression of calreticulin (CRT) and high-mobility group box 1 (HMGB1) which acted as the biomarker of ICD [27]. Images of immunofluorescence staining for cells receiving different treatments are shown in Fig. 4a. An increase in CRT level occurred in CMNP-treated cells, with the high expression of this cytokine acting as an “eat me” signal inducing an immune response. The fluorescence intensity of HMGB1 in cells declined upon treatment with CMNP because the transfer of HMGB1 from the endoplasmic reticulum to the cell surface could trigger an immune response. This released HMGB1 was detected by western blotting (WB) experiments for dual verification (Fig. 4b, c and 4d). The results revealed significantly higher HMGB1 expression in the CMNP group than in the other treatment groups. Apart from ICD, we also conducted research on the cGAS-STING pathway activated by MnO_2_ in macrophages (RAW 264.7). The results of the WB were shown in Fig. S25. The activation of STING was verified in the groups treated with Mn^2+^ (including MnO_2_@BSA/NR, MNP, and CMNP), and the downstream signaling molecule IRF3 (interferon regulatory factor 3) was also active in the above treatment groups. The effect of CMNP of activating immune cells was confirmed, as shown in Fig. 4e. 4T1 cells were cocultured with macrophages (RAW 264.7 cells) for the different treatment groups. Upon treatment with CMNP, the number of M1-type proinflammatory macrophages increased, while the number of M2-type ones decreased [23]. This indicated that anti-tumor macrophages could be activated by CMNP in vitro.Fig. 4Immunogenic cell death of CMNP. The immunofluorescence staining images of (a) calreticulin (CRT) and (b) high mobility group box 1 (HMGB1) after different treatments. Blue marks the nucleus and red fluorescence marks the CRT and HMGB1. (c) Western blotting analysis of HMGB1 expression in 4T1 cells after different treatments. (d) Quantitative analysis of HMGB1 (n = 3). (e) 4T1 cells were co-cultured with macrophages (RAW 264.7 cells) under different treatment groups stimulated. Data are presented as mean ± s.d. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001; compared with PBS group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 4

Biosafety and biodistribution of CMNP

3.4

As CMNP is intended for in vivo applications, its biosafety was rigorously evaluated. To evaluate the biocompatibility of the nanoparticles, the hemolysis induced by them was measured. The hemolysis rate of CMNP was only 3.3% at a concentration of 80 μg/mL, and the hemolysis did not occur during the experiment (Fig. 5a). The biosafety of the CMNP used in vivo was also tested on healthy BALB/c mice. The CMNP was injected intravenously into mice with therapeutic schedule in vivo. Routine blood examinations of the mice were performed, with the results shown in Fig. 5b and S26. The blood biochemical indexes of the mice treated with CMNP were similar to those of the PBS-treated group. Additionally, no clear pathological changes were identified in the hematoxylin-eosin staining (H&E) images of the CMNP-treated mice (Fig. 5c). These results indicated the biosafety of CMNP and its potential for in vivo use. As additional analyses, the tumor-targeting ability and biodistribution of CMNP were determined by in vivo fluorescence imaging of tumor-bearing mice. Cyanine 5.5 (Cy5.5) was used as a fluorescent probe in vivo and loaded into CMNP (Cy5.5-CMNP) and MNP (Cy5.5-MNP). Successful Cy5.5 loading was confirmed via fluorescence spectroscopy, as revealed by the presence of the same excitation and emission peaks in Cy5.5 and Cy5.5-CMNP (Fig. S27). In order to determine the pharmacokinetic comparison data of CMNP and MNP, the Cy5.5-labeled nanoparticles were intravenously injected into healthy mice. The blood drug concentration-time curves of CMNP and MNP were supplied in Fig. S28. The longer circulation of CMNP was detected compared with MNP. The half-life period of CMNP was 7 times higher than MNP (7 h for CMNP and 1 h for MNP). Meanwhile, we also injected Cy5.5-CMNP or Cy5.5-MNP into tumor-bearing mice by intravenously to observe the fluorescence signals at the tumor sites (Fig. 5d). The fluorescence intensity significantly increased from 1 h after Cy5.5-CMNP injection and was maintained at a high level during 72 h of the experiment. Compared with the findings for Cy5.5-MNP, CMNP rapidly accumulated at the tumor site and was retained there for a long time. This favorable tumor-targeting ability of CMNP was attributed to its biomimicry. Particularly, by being coated with a cell membrane, CMNP could effectively penetrate the physiological barriers of the tumor tissues and avoid recognition by the immune system and ensuing phagocytosis. The clearance of CMNP and MNP mainly involved metabolism by the liver and kidneys (Fig. S29). At 72 h after CMNP injection, the levels of Mn and As atoms in the tumor tissues were tested by inductively coupled plasma mass spectrometry (ICP-MS), revealing levels of 2.6 μg/mL and 110.9 ng/mL, respectively. This further confirmed the hypothesis that CMNP could effectively accumulate in tumor tissues (Fig. 5e).Fig. 5The biodistribution and biosafety of CMNP in vivo. (a) Hemolysis and corresponding photographs (inset) of different treatment groups (n = 3). (b) Blood routine examinations including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelet (PLT) of BALB/c mice receiving CMNP treatment, and blood biochemical indexes including aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CR) (n = 3). (c) Hematoxylin and eosin (H&E) staining images of heart, liver, spleen, lung and kidney from different treatment groups. (d) In vivo fluorescence imaging of tumor-bearing mice at different time points after being intravenously injected with Cy5.5-labeled MNP or CMNP. (e) Inductively coupled plasma (ICP) analysis of manganese and arsenic content in tumor tissues (n = 3). Data are presented as mean ± s.d. P > 0.05 (no significance, ns), ∗∗P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 5

Therapeutic effect of CMNP in vivo

3.5

The antitumor effect of CMNP in vivo was evaluated using 4T1 cells from tumor-bearing mice. 4T1 cells in the logarithmic growth phase were subcutaneously inoculated into the back of BALB/c mice (Fig. 6a for the experimental process). The mice were randomly divided into five groups (PBS, DOX, MnO_2_@BSA/NR, MNP, and CMNP). Tumor volume was monitored every day for 10 days, as CMNP-treated tumors entered a stable control phase after 10 days. The tumor growth curve indicated that the tumors treated with PBS grew rapidly. Meanwhile, the groups receiving DOX or MnO_2_@BSA/NR treatment exhibited comparatively low tumor inhibition. The low dose of DOX used in this study was not intended to have a strong anti-tumor effect on its own. Rather than being a stand-alone therapeutic medication, the low-dose DOX was introduced as a synergistic component. Therefore, the limited effectiveness of low-dose DOX monotherapy did not reflect the therapeutic potential of DOX in combination with other agents and was consistent with our experimental design. In order to initiate cascade catalytic reactions in the tumor microenvironment, MnO_2_@BSA/NR depended on its inherent nanozyme activity. This cascade process effectively produced high levels of ROS from endogenous substrates (like H_2_O_2_) in tumors, caused oxidative damage and tumor cell apoptosis.Fig. 6In vivo antitumor efficiency analysis of CMNP. (a) Schematic illustration of the treatment schedule for in vivo antitumor efficiency analysis. (b) Tumor volume growth curves of mice in PBS, DOX, MnO_2_@BSA/NR, MNP, and CMNP treatment groups (n = 5). (c) Corresponding individual tumor volume growth curves of tumor-bearing mice receiving different treatments (n = 5). (d) Ex vivo tumor photographs of tumor-bearing mice receiving different treatments (n = 5). (e) Ex vivo tumor weights of tumor-bearing mice receiving different treatments (n = 5, ∗P < 0.05, compared with PBS group). (f) The survival rates of tumor-bearing mice receiving different treatments (n = 5). (g) Representative hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and hypoxia inducible factor 1 subunit alpha (HIF-1α) staining images of tumor tissues with different treatments. (h) Western blotting analysis of GPX4 expression in tumor tissues after different treatments. (i) Quantitative analysis of GPX4 corresponding to (h) (n = 3, ∗∗P < 0.01, ∗∗∗P < 0.001; compared with PBS group). (j) Western blotting analysis of IRF3, p-IRF3, STING, and p-STING expression in tumor tissues after different treatments. (k) Quantitative analysis of p-STING/STING corresponding to (j) (n = 3, P > 0.05, no significance, ns, ∗∗P < 0.01, ∗∗∗P < 0.001; compared with CMNP group). (l) Quantitative analysis of p-IRF3/IRF3 corresponding to (k) (n = 3, ∗P < 0.05, ∗∗∗P < 0.001; compared with CMNP group). Data are presented as mean ± s.d.Fig. 6

In contrast, the MNP group demonstrated better tumor inhibition, which may be attributable to the combined therapy. The primary distinction between MNP and MnO_2_@BSA/NR was the integrated design of MNP, which combined low-dose DOX to create a dual-pathway therapeutic system while maintaining the nanozyme cascade ROS production function of MnO_2_@BSA/NR. In particular, the ROS produced by the MnO_2_@BSA/NR component caused intense oxidative stress in tumor cells, which could make tumor cells more vulnerable to DOX-mediated DNA damage [36,37]. Meanwhile, DOX-induced DNA damage could exacerbate the oxidative stress-induced apoptotic response in tumor cells. Compared to MnO_2_@BSA/NR, which solely depended on one ROS-mediated pathway, MNP exhibited stronger anti-tumor activity due to the cascade synergistic effect between the two components. Low-dose DOX by itself was not very effective, but when combined with MnO_2_@BSA/NR in MNP, it produced a 1 + 1 > 2 therapeutic effects. Notably, the CMNP treatment group exhibited the best antitumor efficiency. This may be attributable to the unique targeting properties conferred by the tumor cell membrane envelope on the CMNP surface. These properties enabled more CMNP nanoparticles to reach the tumor site, thereby enhancing tumor cell killing (Fig. 6b and c). At the end of the experiment, the tumor tissues were collected, photographed, and weighed. Upon observing and weighing these tissues, the results were consistent with the tumor growth curve, confirming the antitumor effect of each treatment (Fig. 6d and e). Mouse weight was also monitored throughout the treatment with the various drugs. The results demonstrated negligible changes in body weight, which supports the excellent biosafety of CMNP and suggests that it does not substantially interfere with mouse health (Fig. S30).

As expected, the superior therapeutic efficiency of CMNP significantly prolonged the survival of the mice. Upon survival analysis, it was determined that 40% of the mice survived until the 55th day, while survival was relatively short in the PBS group. Indeed, the mean survival time of the CMNP group was twice that of the PBS group, highlighting the therapeutic effect of CMNP on tumor development (Fig. 6f). The H&E, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and hypoxia inducible factor 1 subunit alpha (HIF-1α) images of tumor tissues with different treatment groups were shown in Fig. 6g. The most cell apoptosis and necrosis of tumor were tested after CMNP treatment. This higher tumor apoptosis and necrosis were probably attributed to ferroptosis and immune activation. As shown in the in vitro experiments, owing to the large amount of ROS produced by CMNP, an enhanced rate of ferroptosis was observed in the tumor cells. To quantify this, we determined the glutathione peroxidase 4 (GPX4) level by WB. This level decreased significantly in the MNP and CMNP treatment groups, with the lowest level being found upon CMNP treatment. The high ferroptosis activity effectively suppressed tumor growth (Fig. 6h and i). Besides the unbalanced redox, immune activation also enhanced the antitumor effect. The activation of STING was verified in the tumor, and IRF3 as a downstream signal molecule of the cGAS-STING pathway was active in the groups treated with CMNP (Fig. 6j, k and 6l). Zhang and collaborators have demonstrated in earlier research how transition metal oxide nanozymes can activate the cGAS-STING pathway for improved tumor immunotherapy [[38], [39], [40]]. In particular, we study oxide and sulfide nanozyme cascade catalysis. This strategy aims to strengthen the synergistic integration of immunotherapeutic techniques with nanozymes.

Although cGAS-STING pathway activation was confirmed, there was also a need to analyze the tumor-killing effect by determining the cell-mediated immune response. Therefore, we analyzed the proportions of immune cells and corresponding secretion levels of cytokines using flow cytometry and enzyme-linked immunosorbent assay (ELISA). First, phenotypic analysis of immune cells in spleen, peripheral blood and tumor tissues was conducted using flow cytometry. CD8^+^ T cells (cytotoxic T lymphocytes, CTLs) are the core effector cells of the antitumor immune response [41]. CD4^+^ T cells assist in activating B cells and CD8^+^ T cells by secreting cytokines [42]. The proportion of CD4^+^ T and CD8^+^ T cells in spleen, peripheral blood and tumor was higher in CMNP than PBS treatment group (Fig. 7a, b, 7c, 7d, 7e, 7f and S31). The flow cytometry analysis of T cells in the spleen showed that, compared with the saline group, the CD8^+^ T and CD4^+^ T cells increased by 176% and 135% respectively after CMNP treatment. Similar changes were also observed in the peripheral blood. After CMNP treatment, the number of CD8^+^ T cell and CD4^+^ T cells increased by 149% and 148% respectively, indicating that CMNP can induce the activation of T cells. Especially the proportion of CD4^+^ T cells was 40.8%, which was significantly higher than the rate in the PBS group in tumor (23.6%) (Fig. S32). Moreover, the proportion of CD8^+^ T cells in the CMNP group was 26.7% ± 6.3%, which was approximately 4.1 times higher than that in the PBS group 6.5% ± 1.3% in tumor (P < 0.01) (Fig. S33). These results indicated that the CMNP could active T cells in spleen, and these cells were transported by peripheral blood and recruited in tumor. In the tumor microenvironment, the phenotypic polarization of macrophages has a significant impact on tumor progression. M1-type macrophages have proinflammatory and antitumor activities, while M2-type ones exhibit immunosuppressive and protumor characteristics [43]. Further macrophage phenotype analysis in tumor tissues showed that the proportion of M1 (34.5%) macrophages (F4/80^+^CD86^+^) significantly increased after CMNP treatment, being 4.9 times higher than that in the PBS group (Fig. 7g and h). Correspondingly, the proportion of M2 macrophages (F4/80^+^CD206^+^) in tumor decreased significantly after CMNP treatment (4.9%) compared with that in the PBS group (38.3%) (Fig. 7i, j and S34), resulting in a 48-fold increase of the M1:M2 ratio compared with that in the PBS group (Fig. S35). The M1-typed polarization of macrophages in spleen was also detected in Fig. S36 and the slightly increase of the M1:M2 ratio with CMNP treated groups than in PBS group (Fig. S37). We used ELISA to determine the content of proinflammatory cytokines (TNF-α, IFN-γ, and IL-2) in the tumor tissues. The levels of these cytokines are important indicators of antitumor immunity [44]. The results showed that the expression of the cytokines TNF-α, IFN-γ, and IL-2 in the CMNP treatment group was significantly higher than that in the other treatment groups. Compared with the findings in the PBS group, the levels of IFN-γ and TNF-α secretion were increased by 2.1-fold and 2.2-fold, respectively (P < 0.001), which is highly consistent with the mechanism of Mn^2+^ activating the cGAS-STING pathway. Moreover, the increased secretion of IL-2 suggested that CMNP could promote T-cell proliferation and activation (Fig. 7k, l and 7m). The aforementioned findings suggested that the tumor suppressive mechanism of CMNP results from a positive feedback regulation driven by ROS generation and immune activation. Consequently, we elucidated the potential mechanism of the CMNP-mediated amplification of the immunotherapeutic cascade and the promotion of apoptosis (Fig. 7n). CMNP generated oxygen via endogenous H_2_O_2_ depletion, thus alleviating hypoxia in the tumor microenvironment. The produced O_2_ could also promote the switching of the macrophage phenotype to the M1 type, which synergistically enhanced the effect of immunotherapy. The chemotherapeutic agent doxorubicin (DOX) induced ICD of tumor cells, while Mn^2+^ activated the cGAS-STING pathway to stimulate systemic immune responses. The synergistic interplay among these components within CMNP collectively achieved potent antitumor effects.Fig. 7In vivo immune activation analysis of CMNP. Representative flow cytometry analysis of CD4^+^ T cells (CD3^+^CD4^+^) in (a) peripheral blood, (b) spleen and (c) tumor tissues. Representative flow cytometry analysis of CD8^+^ T cells (CD3^+^CD8^+^) in (d) peripheral blood, (e) spleen and (f) tumor tissues. (g) Representative flow cytometry analysis of M1 macrophages (F4/80^+^CD86^+^) in tumor tissues. (h) Corresponding quantification of M1 macrophages (F4/80^+^CD86^+^) in tumor tissues (n = 3). (i) Representative flow cytometry analysis of M2 macrophages (F4/80^+^CD206^+^) in tumor tissues. (j) Corresponding quantification of M2 macrophages (F4/80^+^CD206^+^) in tumor tissues (n = 3). Quantififications of (k) interleukin-2 (IL-2), (l) tumor necrosis factor-α (TNF-α), and (m) interferon-γ (IFN-γ) in tumor tissues (n = 3). (n) Schematic illustration of the mechanism of immunotherapeutic. Data are presented as mean ± s.d. P > 0.05 (no significance, ns), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001; compared with PBS group.Fig. 7

Conclusion

4

In this study, we have constructed a cell-membrane coated biomimetic nanozyme platform (CMNP) that addresses critical challenges in nanocatalytic tumor therapy. By integrating manganese dioxide nanoparticles (MnO_2_@BSA), nano-realgar (NR), and doxorubicin (DOX) with tumor cell membrane biomimetics, CMNP achieves tumor targeting and multi-mechanistic therapeutic effects. The MnO_2_ decomposes endogenous hydrogen peroxide to alleviate tumor hypoxia, creating a microenvironment conducive to enhancing NR's glutathione oxidase- and L-cysteine oxidase-like activities, which generate cytotoxic reactive oxygen species (ROS) to induce tumor apoptosis. Meanwhile, the released Mn^2+^ ions activate the cGAS-STING signaling pathway, orchestrating an immune response that recruits cytotoxic immune cells into the tumor microenvironment. This synergistic interplay between nanocatalytic ROS generation and immune activation establishes a robust dual-pronged strategy to amplify tumor suppressive effects. Our findings demonstrate that this versatile nano-platform not only overcomes limitations of traditional nanocatalytic systems but also integrates catalytic therapy with immunomodulation, offering a promising paradigm for developing next-generation cancer treatments. The utilization of nano-realgar, a nanocrystallized form of traditional Chinese medicine, further highlights the potential for interdisciplinary innovation in nanomedicine, providing a novel and valuable approach for precision tumor therapy.

CRediT authorship contribution statement

Chen Bai: Supervision, Writing – original draft. Peng Hu: Data curation, Methodology. Zhongmin Ni: Writing – original draft. Jun Xie: Methodology, Resources. Fang Cai: Methodology. Jiale Wang: Methodology. Xianbin Wang: Methodology. Dong Guo: Writing – review & editing.

Declaration of competing interest

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