A calcium manganese-based pancatalytic nanozyme as a cell pyroptosis inhibitor for efficient inflammatory bowel disease treatment

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder marked by dysregulated immune responses and pyroptosis of intestinal epithelial cells, a type of programmed cell death that aggravates inflammation. Current therapeutic strategies frequently encounter limitations in both efficacy and specificity. Herein, we developed a calcium manganese-based (CaMn₂O₄) pancatalytic nanozyme designed to mitigate pyroptosis and alleviate IBD symptoms. The calcium manganese-based nanozyme exhibits dual enzyme (catalase and superoxide dismutase)-mimetic catalytic activities, scavenging reactive oxygen species and suppressing GSDMD cleavage, a key mediator of pyroptosis. In vitro studies demonstrated that the CaMn₂O₄ nanozyme significantly reduced pyroptotic cell death in lipopolysaccharide (LPS)/nigericin (Ni)-stimulated cells. In a murine colitis model, CaMn₂O₄-based nanozyme treatment attenuated inflammatory infiltration, preserved epithelial barrier integrity, and downregulated pyroptosis-related markers. This study highlights the potential of pancatalytic nanotherapy targeting pyroptosis as a novel strategy for IBD treatment.

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Meiqi Chang

A/Prof. Meiqi Chang received her PhD degree in Chemistry from Jilin University in 2019. She is now an associate researcher at Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine. Her current research interests include the design and preparation of multifunctional nanomaterials and nanozymes for regulation of cell pyroptosis.

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Introduction

Inflammatory bowel disease (IBD), primarily encompassing Crohn's disease and ulcerative colitis, is a chronic, relapsing inflammatory disorder affecting the gastrointestinal tract that affects millions of patients worldwide.^1,2 The pathogenesis of IBD involves genetic susceptibility, environmental factors, and the resulting dysregulated immune response, ultimately leading to persistent mucosal inflammation, oxidative stress, and epithelial barrier dysfunction.^3,4 Despite significant advances in the use of biologic therapies (such as anti-TNF-α drugs and IL-12/IL-23 inhibitors), a significant proportion of patients still experience primary nonresponse or secondary loss of efficacy (secondary nonresponse).^5–7 This situation highlights the urgent need for novel therapeutic strategies targeting alternative inflammatory pathways.

Recent studies have implicated pyroptosis – a highly inflammatory form of programmed cell death – as a critical driver of IBD progression.⁸ Pyroptosis is mediated by gasdermin family proteins (e.g., GSDMD), which form plasma membrane pores upon cleavage by inflammatory caspases (e.g., caspase-1/4/5/11), facilitating the release of pro-inflammatory cytokines (IL-1β, IL-18) and cellular contents.^9,10 The activation of pyroptosis is primarily regulated by the NLRP3 inflammasome, a multiprotein complex that detects cellular damage and pathogen-associated molecular patterns.^11,12 A recent review by Liu et al. provides a comprehensive overview of the molecular mechanisms of pyroptosis and its role in various diseases, including IBD.¹³ Excessive NLRP3 inflammasome activation aggravates mucosal injury by promoting pyroptosis in intestinal epithelial cells (IECs) and immune cells, perpetuating a vicious cycle of inflammation and tissue damage.^14,15 Another study by Fang et al. discusses the emerging mechanisms of pyroptosis in inflammatory diseases, highlighting its potential as a therapeutic target.¹⁶ Current therapies rarely address pyroptosis directly, leaving a critical therapeutic gap.

Nanocatalytic therapy has emerged as a promising strategy for inflammatory diseases by leveraging nanozymes to modulate redox and immune homeostasis.^17,18 Especially, the emerging pancatalytic medicine involves the holistic control of the preparation (P) of a catalyst, activation (A) of the specific biological effect and non-toxic (N) treatment of versatile diseases, by which an efficient therapeutic outcome by pancatalytic treatment can be achieved.¹⁹ Recent studies have highlighted the potential of nanozymes in treating IBD by reducing oxidative stress and inhibiting pro-inflammatory cytokines. For example, a study by Lei et al. reviewed the recent advancements in nanozyme-based materials for IBD, emphasizing the importance of structural design and therapeutic strategies.²⁰ Another study demonstrated the therapeutic efficacy of MPBZs in a DSS-induced mouse model of colitis, showing significant reductions in pro-inflammatory cytokines and oxidative stress markers.²¹ Unlike conventional drugs, nanozymes exhibit enzyme-like activities (e.g., superoxide dismutase (SOD) and catalase (CAT)) that scavenge reactive oxygen species (ROS) and suppress oxidative stress, a key trigger for NLRP3 inflammasome activation.^15,22,23 Among potential candidates, calcium and manganese are biologically essential elements with distinct roles in inflammation regulation. Calcium signaling is pivotal for maintaining epithelial barrier integrity and immune cell function, while manganese ions (Mn²⁺) serve as antioxidant nanoenzymes for scavenging ROS.^24,25 However, the synergistic combination of Ca²⁺ and Mn²⁺ in a nanocatalytic system for pyroptosis inhibition has not been explored, presenting a unique opportunity to develop an intelligent IBD therapy.

Herein, we designed a calcium manganese-based (CaMn₂O₄) pancatalytic nanozyme (abbreviated as the CaMn nanozyme) to disrupt pyroptosis and mitigate IBD progression (Scheme 1). The pancatalytic IBD treatment involved the initial precise preparation and characterization of the CaMn nanozyme. In the activation of the biological effect, the CaMn nanozyme could scavenge ROS via SOD/CAT-mimetic activity, reduce oxidative stress and inhibit GSDMD cleavage, thereby blocking pyroptotic cell death. In vitro experiments showed that the CaMn nanozyme effectively suppressed pyroptotic cell death in lipopolysaccharide (LPS)/nigericin (Ni)-treated cells. In the final IBD treatment on a mouse colitis model, CaMn nanozyme therapy reduced inflammatory infiltration, maintained epithelial barrier function, and decreased pyroptosis-related biomarkers. This study introduces a CaMn-based nanozyme for pyroptosis inhibition in IBD, bridging the gap between nanomedicine and inflammasome biology. Our findings highlight the potential of using redox-modulating nanozymes as next-generation therapeutics for inflammatory disease.

Scheme 1 Schematic diagram of the synthesis of CaMn₂O₄(CaMn) nanozyme and its application mechanism in inflammatory bowel disease. (a) Synthesis process of CaMn nanozyme. (b) Mechanism diagram of CaMn nanozyme in inflammatory bowel disease treatment. CaMn nanozyme effectively inhibits cellular pyroptosis by scavenging excessive ROS, demonstrating synergistic anti-inflammatory and anti-cellular pyroptosis therapeutic effects.

Experimental

Materials

Calcium chloride (CaCl₂, 96%), manganese chloride (MnCl₂, 99%), potassium permanganate (KMnO₄, 99.5%) and potassium hydroxide (KOH, 85%) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), 2,2′-zino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and potassium persulfate were purchased from Aladdin (Shanghai, China). Cell counting kit-8 (CCK-8) and the Calcein AM/PI cell viability/cytotoxicity assay kit were obtained from Yeasen Biotechnology (Shanghai, China). The human IL-1β ELISA kit was provided by Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China). 2′,7′- dichlorofluorescein diacetate (DCFH-DA) and 4′,6′-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). McCoy's 5A medium, fetal bovine serum (FBS), and 1% penicillin/streptomycin were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Anti-N-GSDMD, anti-GSDMD and anti-ACTIN antibodies were purchased from Abcam. LPS was obtained from Sigma-Aldrich (California, USA). Rhodamine B (RhB), Ni and 5-aminosalicylic acid (5-ASA) were purchased from MedChemExpress (New Jersey, USA). Dextran sodium sulfate (DSS, M_w 36 000–50 000 Da) was obtained from MP Biomedicals (California, USA). Paraformaldehyde (PFA) was purchased from Servicebio (Wuhan, China).

Synthesis of CaMn

CaMn was synthesized using a hydrothermal method. The specific operation is as follows: 1 mM CaCl₂, 0.6 mM KMnO₄ and 1.4 mM MnCl₂ were dissolved in 40 mL deionized water under magnetic stirring. Next, KOH solution at a concentration of 7.8 M in a volume of 10 mL was added dropwise to the above mixed solution. Subsequently, the obtained mixture was transferred to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene and heated at a constant temperature of 180 °C for 72 hours. The resulting brown solid was then centrifuged and washed several times with distilled water to remove impurities. Finally, the washed brown solid was placed in a vacuum oven at 60 °C and dried overnight to obtain the product.

For in vitro and in vivo experiments, BSA modification was applied for improvement of stability and dispersibility: 10 mg product was dissolved in 10 mL deionized water, then 100 mg BSA was added and stirred for 24 hours. Finally, the product was washed sequentially with water and ethanol and then centrifuged to obtain CaMn.

Characterization of the CaMn nanozyme

The morphology of the CaMn nanozyme was observed using a JEM-2100F transmission electron microscope (TEM). Elemental mapping images and energy dispersive spectroscopy (EDS) line scan measurements were employed to verify the distribution of elements (JEOL-F200, Japan). X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of CaMn₂O₄ nanocatalysts (Thermo Scientific K-Alpha, USA). X-ray diffraction (XRD) measurement was obtained with a diffractometer (Rigaku SmartLab SE, Japan). The sample absorbance was measured using a UV-vis-NIR spectrometer (Shimadzu, Japan). Fluorescence was imaged using a Carl Zeiss LSM710 fluorescence microscope. Confocal images were obtained by observation with a confocal laser scanning microscope (Carl Zeiss, Germany).

RhB-coupled CaMn nanozyme

20 μg of RhB was dissolved in 10 mL deionized water, then 10 mg CaMn nanozyme was added and stirred for 24 hours. Finally, the final product was collected by centrifugation and washed with deionized water to remove excess RhB.

ABTS radical scavenging

The ability of ABTS radical cation radicals (ABTS+˙) was measured using the ABTS radical cation decolorization method. ABTS solution (7.4 mM L⁻¹) and potassium persulfate solution (2.6 mM L⁻¹) were mixed in equal volumes and reacted in the dark for 24 hours to produce ABTS+˙. The absorbance at 734 nm was measured for mixtures, where ABTS+˙ was reacted with varying concentrations of the CaMn nanozyme for 5 min or with a constant concentration of the CaMn nanozyme for different time intervals. All experiments were performed in triplicate.

DPPH radical scavenging

The ability of DPPH radicals (DPPH˙) was measured using the DPPH radical cation decolorization method. An ethanol solution of DPPH (0.04 mg mL⁻¹) was prepared, and its absorbance at 517 nm was measured after reacting with varying concentrations of the CaMn nanozyme for 30 min or with a fixed concentration of the CaMn nanozyme for different time intervals. All experiments were performed in triplicate.

Cell cultures and pyroptosis model induction

Human colonic epithelial cell line HT-29 was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. The cells were cultured in McCoy's 5A medium supplemented with 10% (v/v) FBS, and 1% penicillin/streptomycin at 37 °C with 5% carbon dioxide (CO₂). To establish the pyroptosis model, HT-29 cells were first stimulated with LPS (1 μg mL⁻¹) for 4 hours, followed by the addition of Ni (3 μM) for another 2 hours, as previously described.²⁶ This method was adapted to our experimental conditions to ensure optimal induction of pyroptosis in our cell system.

In vitro cellular uptake assay

HT-29 cells were plated in 24-well plates at a density of 5 × 10⁴ cells per well and incubated overnight at 37 °C. The medium was replaced with either fresh McCoy's 5A or the RhB-coupled CaMn nanozyme at predetermined time points and incubated at 37 °C with 5% CO₂. After washing three times with PBS, the cells were stained with DAPI for 10 minutes and imaged using a confocal laser scanning microscope (CLSM).

CCK-8

HT-29 cells were plated in 96-well plates at a density of 1 × 10⁴ cells per well and incubated overnight at 37 °C. To assess the cytotoxicity of the CaMn nanozyme, the cells were treated with predetermined concentrations of the compound for 6 hours. Subsequently, the CCK-8 reagent diluted in a culture medium at a ratio of 1 : 10 was added to each well, and the cells were incubated for an additional hour. The absorbance at 450 nm was measured using SpectraMax iD5.

Western blot

After digestion with lysis buffer containing protease inhibitors for 30 minutes on ice, the protein solution was collected and quantified using a BCA protein assay kit. Subsequently, 30 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature. The membrane was incubated with the anti-N-GSDMD antibody (1 : 1000) and the anti-ACTIN antibody (1 : 2000) overnight at 4 °C. After washing three times with TBST, the membrane was incubated with corresponding secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature. The membrane was then imaged using an enhanced chemiluminescence (ECL) system and quantified using Image J software.

Integrated cell-based assays

HT-29 cells were plated in 24-well plates at a density of 5 × 10⁴ cells per well and incubated overnight at 37 °C. The cells were then treated as follows: (1) control group; (2) LPS + Ni group: cells were stimulated with LPS (1 μg mL⁻¹) for 4 hours, Ni (3 μM) was added and incubated for another 2 hours; and (3) LPS + Ni@CaMn group: cells were co-treated with the CaMn nanozyme (100 μg mL⁻¹) and LPS (1 μg mL⁻¹) for 4 hours, Ni (3 μM) was added and incubated for another 2 hours.

(i) The cell supernatant was collected and the IL-1β level was determined using an ELISA kit, (ii) morphological changes were imaged using a microscope at 4–6 hours, (iii) intracellular ROS were quantified with DCFH-DA, and (iv) live/dead assay was evaluated with Calcein-AM/PI staining followed by imaging using a Carl Zeiss LSM710 fluorescence microscope.

Mouse colitis model

C57BL/6 mice (male, 6 weeks old) were purchased from GemPharmatech Co. Ltd and feed at the Laboratory Animal Center of Shanghai Tenth People's Hospital. All animal experiments followed the requirements of the Guidelines for the Care and Use of Laboratory Animals issued by the National Research Council and were approved by the Animal Ethics Committee (IACUC) of Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine (Number: 2025110). Mice were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and had free access to standard mouse chow and water. To induce colitis, C57BL/6 mice were treated with 3% DSS in autoclaved drinking water for 8 days.

In vivo biocompatibility assay

Six-week-old male C57BL/6 mice were selected for the study. The experimental group received a daily oral gavage of the CaMn nanozyme at a dose of 12 mg kg⁻¹, while the control group was administered an equal volume of PBS. This treatment regimen was maintained for 8 consecutive days. Throughout the experiment, the body weight of the mice was monitored daily. On the 9th day, the mice were anesthetized and sacrificed, and blood samples were collected for routine blood tests and biochemical analyses. Concurrently, the digestive system and major organs (heart, liver, spleen, lung, and kidneys) were collected. These tissues were fixed in PFA, processed into paraffin sections, and subjected to hematoxylin–eosin (H&E) staining for histopathological evaluation.

Evaluation of therapeutic efficacy of colitis in mice

C57BL/6 mice were randomly divided into four groups (n = 5): (1) Control; (2) DSS; (3) DSS@CaMn (12 mg kg⁻¹); and (4) DSS@5-ASA (50 mg kg⁻¹). Except for the control group, the other groups were given 3% DSS solution (dissolved in sterile drinking water) for 8 consecutive days to establish an acute colitis model. The control and DSS groups were treated with 0.2 mL PBS, and the other groups were administered the CaMn nanozyme (12 mg kg⁻¹) or 5-ASA (50 mg kg⁻¹) on predetermined days. The mice weight changes were monitored and sacrificed on day 9. At the same time, the colon length was measured, the intestinal tissues were collected and fixed with 4% PFA, and paraffin sections were prepared for H&E staining and immunohistochemical staining to evaluate pathological damage.

H&E staining

The intestinal tissues were fixed in 4% PFA for 24 hours, dehydrated with graded ethanol, transparentized with xylene and embedded in paraffin. Serial sections of 4 μm thickness were prepared using a rotary microtome. After drying at 60 °C overnight, they were stained using a standard H&E staining procedure.

Immunohistochemical staining

Briefly speaking, the intestinal tissues were fixed in 4% PFA and embedded in paraffin. After cutting into sections, the tissues were incubated with primary antibodies overnight at 4 °C, followed by appropriate secondary antibodies. Finally, the results of images were photographed using a scanner.

Statistical analysis

All data were analyzed using GraphPad Prism software. Data are presented as mean ± standard deviation (s.d.). Statistical results were analyzed using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant.

Results and discussion

Synthesis and characterization of the CaMn nanozyme

The CaMn nanozyme was synthesized using a hydrothermal method. Transmission electron microscopy (TEM) images show the nanowire morphology (Fig. 1a). The characteristic signals and homogeneous distributions of Ca, O, and Mn detected in the X-ray energy dispersive spectrum mapping profile confirmed the successful synthesis of the CaMn nanozyme (Fig. 1b and c). The crystal structure of the CaMn nanozyme was confirmed by X-ray diffraction analysis (JCPDS 76-0516) (Fig. 1d). A representative ball-and-stick model of the CaMn nanozyme is presented in Fig. 1e. The survey X-ray photoelectron spectrum (XPS) indicates the coexistence of Ca, Mn and O (Fig. 1f). In the high-resolution spectrum of Ca 2p, the peaks at 348.4 eV and 344.8 eV are assigned to Ca 2p_1/2 and Ca 2p_3/2, respectively (Fig. 1g). In the high-resolution spectrum of Mn, the peaks at 652.7 eV and 641.1 eV correspond to the presence of Mn³⁺, while the peaks at 643.5 eV and 656.3 eV correspond to the presence of Mn⁴⁺, respectively (Fig. 1h). Three characteristic peaks at 531.3 eV, 530.1 eV and 528.5 eV in the O 1s spectrum could be attributed to the surface-adsorbed oxygen, oxygen vacancies and lattice oxygen (Fig. 1i).

Fig. 1 Characterization of CaMn nanozyme. (a) TEM and (b) elemental mapping images of CaMn nanozyme. (c) EDS spectral analysis of CaMn nanozyme. (d) X-ray diffraction pattern of CaMn nanozyme. (e) Schematic diagram of crystal structure models of CaMn nanozyme. XPS spectra of (f) wide-scan (g) Ca 2p, (h) Mn 2p, and (i) O 1s of CaMn nanozyme.

ROS scavenging activity of the CaMn nanozyme

The absorption of different concentrations of the CaMn nanozyme was monitored by UV-Vis spectroscopy, and the absorption intensity increases with the increase of the concentration (Fig. 2a and Fig. S1). To comprehensively evaluate the antioxidant capability of the CaMn nanozyme, we tested the scavenging ability of the CaMn nanozyme toward radicals. The highly stable radical cation ABTS+˙ is generated by the oxidation of ABTS with potassium sulfite. The total antioxidant capacity of the CaMn nanozyme was accessed by monitoring its absorption changes in the visible band. Firstly, concentration-dependent ABTS free radical scavenging capacity of the CaMn nanozyme was monitored via UV-Vis spectroscopy, and the radical scavenging efficiency reaches 84.7% when the concentration is 200 μg mL⁻¹ (Fig. 2b, c and Fig. S2). Moreover, ABTS free radical scavenging experiments of the CaMn nanozyme indicate time-dependent features (Fig. 2d, e and Fig. S3). In addition, 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH˙) are the nitrogen centered radicals widely used for the determination of total antioxidant capacity of natural antioxidants. Similarly, DPPH free radical scavenging experiments exhibit concentration-dependent (Fig. 2f and g) and time-dependent characteristics (Fig. 2h, i and Fig. S4). The radical scavenging efficiency can reach up to 41.6%.

Fig. 2 Free-radical-scavenging capabilities of CaMn nanozyme. (a) The absorbance of different concentrations of CaMn nanozyme was monitored by UV-Vis spectroscopy. Concentration-dependent ABTS free radical scavenging capacity of CaMn nanozyme monitored via (b) UV-Vis spectroscopy and (c) corresponding quantitative analysis (n = 3). Time-dependent ABTS free radical scavenging capacity of CaMn nanozyme monitored via (d) UV-Vis spectroscopy and (e) corresponding quantitative analysis (n = 3). Concentration-dependent DPPH scavenging capacity of CaMn nanozyme monitored via (f) UV-Vis spectroscopy and (g) corresponding quantitative analysis (n = 3). Time-dependent DPPH scavenging capacity of CaMn nanozyme monitored via (h) UV-Vis spectroscopy and (i) corresponding quantitative analysis (n = 3).

In vitro therapeutic efficacy and evaluation of pyroptosis inhibition

Considering that the CaMn nanozyme exhibits remarkable antioxidase-mimetic activities, we then systematically examine its application potential for ROS scavenging and the specific mechanism in vitro. The time-dependent uptake process of the CaMn nanozyme was evaluated using a confocal laser scanning microscope (CLSM) (Fig. 3a). The enhanced red fluorescence intensity of the Rhodamine B-labelled CaMn nanozyme confirmed the occurrence of the internalization process. Cytotoxicity of the CaMn nanozyme toward HT-29 cells with different concentrations was measured. The negligible cytotoxicity confirms the excellent biocompatibility (Fig. 3b). Considering the pro-inflammatory properties of this disease, we first investigated the anti-pyroptosis ability of the CaMn nanozyme. Western blot analysis and affiliated quantitative analysis of N-GSDMD were initiated to assess protein expression. Elevated expression of N-GSDMD proteins was observed in the LPS/Ni group, underpinning the permeabilization of the plasma membrane and the formation of membrane pores. The treatment group reduced the expression of N-GSDMD (Fig. 3c, d and Fig. S5). Meanwhile, the CaMn nanozyme significantly reduced the level of the pro-inflammatory cytokine IL-1β (Fig. S6). Cell morphology observation was initially introduced as the most intuitive method. Cells treated with the LPS/Ni group exhibited swelling features with the appearance of large bubbles, which distinguished them from the control group, confirming the occurrence of pyroptosis. It is worth noting that intervention of the CaMn nanozyme inhibits the occurrence of cell pyroptosis (Fig. 3e). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) is applied as the fluorescence probe to explore intracellular ROS levels. Different from the LPS/Ni group with bright green fluorescence, the addition of the CaMn nanozyme greatly reduced the fluorescence intensity, which confirms the ROS scavenging ability of the CaMn nanozyme (Fig. 3f). To visualize cell viability, the Calcein-AM/PI probe was applied to stain the HT-29 cells. It was observed that LPS/Nigericin treatment effectively killed a substantial proportion of the colon cancer cells, as reflected by the cell-stained magenta by the PI dye (Fig. 3g).

Fig. 3 In vitro efficacy assessment of CaMn nanozyme in inflammatory bowel disease. (a) CLSM images of HT-29 cells treated with RhB-labeled CaMn nanozyme after incubation for 0, 2 and 4 h. The blue channel showed nuclei stained with DAPI. The red channel showed CaMn nanozyme labeled with RhB (scale bar: 10 μm). (b) Cytotoxicity of CaMn nanozyme toward HT-29 cells with different concentrations (n = 5). (c) Western blot analysis of N-GSDMD in HT-29 cells after varied treatments. (d) The corresponding quantitative analysis of N-GSDMD protein expressions based on western blotting results (n = 3). (e) Morphological features of HT-29 cells with different treatments, as revealed by bright field scanning confocal images (scale bar: 5 μm). Red arrows represent cell swelling with big bubbles. (f) Intracellular ROS level of HT-29 cells after different treatments (scale bar: 100 μm). (g) Calcein AM/PI staining of HT-29 cells after different treatments (scale bar: 200 μm).

In vivo assessment

The verification of the biological safety of the CaMn nanozyme is necessary before its application in animal experiments (Fig. 4a). The nanomedicine formulation demonstrated excellent biocompatibility profiles, as evidenced by stable body weight, and normal ranges in both biochemical and hematological parameters (Fig. 4b–f and Fig. S7). Histopathological examination (H&E staining) of major organs including the heart, liver, spleen, lung, kidney, stomach, duodenum, jejunum, ileum, proximal colon and distal colon revealed a normal histological architecture without detectable pathological alterations, demonstrating the nanomedicine's excellent biosafety profile (Fig. 4g and h).

Fig. 4 Biosafety of CaMn nanozyme in vivo. (a) Schematic diagram of the experimental protocol for biosafety. (b) Body weight change after treatment with CaMn nanozyme or PBS (n = 5). Biochemical parameters of the mouse after treatment with CaMn nanozyme or PBS, including (c) alanine aminotransferase (ALT), (d) ceramic aspartate aminotransferase (AST), (e) creatinine (CRE), (f) blood urea nitrogen (BUN) (n = 5). All data are shown as the mean ± standard deviation (s. d.). (g) H&E staining of heart, liver, kidney, lung, and spleen after treatment with CaMn nanozyme or PBS for 8 days (scale bar: 100 μm). (h) H&E staining of the digestive tract, including stomach, duodenum, jejunum, ileum, proximal colon and distal colon (scale bar: 100 μm).

Based on the demonstrated efficacy of the CaMn nanozyme in ROS scavenging and pyroptosis inhibition, an in vivo assessment was conducted. To establish an acute IBD model, mice were subjected to 8-day oral administration of 3% DSS in their water supply (Fig. 5a). To evaluate the protective effects against colitis, mice were orally given nanomedicines daily from day 0 to 8. A disease control group (IBD mice treated with PBS) and 5-ASA (the gold standard for IBD) were included for comparison. Fig. 5b demonstrates that mice in the non-DSS group displayed normal physiological activity and consistent body weight, in contrast to DSS-treated control mice, which suffered from a pronounced weight reduction along with clinical signs of diarrhea and hematochezia. Therapeutic efficacy was evidenced by marked symptom alleviation and reduced weight loss following nanomedicine and 5-ASA treatment (Fig. S8). Gastrointestinal bleeding can lead to anemia, which is usually accompanied by significant spleen enlargement. In the DSS-induced IBD model, we observed a significant enlargement of the spleen. In contrast, the spleens of mice in the CaMn nanozyme- or 5-ASA-treated groups did not change significantly compared with the control group, suggesting that anemia induced by gastrointestinal bleeding did not occur (Fig. S9). Moreover, CaMn nanozyme treatment significantly attenuated mitigated colonic tissue injury, as evidenced by preserved colon length (Fig. 5c and d). Compared to untreated IBD controls demonstrating characteristic colon pathology including deep ulcerations, epithelial destruction, and leukocyte infiltration, CaMn nanozyme-administered groups exhibited marked tissue repair (Fig. 5e). GSDMD, an indicator of pyroptosis, was assessed to further confirm the therapeutic effect of nanomaterials (Fig. 5f), and the reduced expression of GSDMD in the DSS group confirmed the occurrence of pyroptosis. The intervention of nanomedicine significantly inhibited the development of pyroptosis.

Fig. 5 Efficacy assessment of CaMn nanozyme for inflammatory bowel disease. (a) Schematic diagram of the experimental protocol for mouse therapeutic evaluation. (b) Body weight change with various treatments (n = 5). All data are shown as the mean ± standard deviation (s. d.). (c) Digital photographs of colon tissue after different treatments. (d) Colon length was measured and analyzed (n = 5; ****P < 0.0001). Data are presented as mean ± standard deviation (s. d.). (e) H&E staining images of colon tissue with various treatments (scale bar, 100 μm). (f) Representative images of GSDMD staining with various treatments (scale bar, 100 μm or 1 mm).

Conclusion

In summary, we developed a calcium manganese-based pancatalytic nanozyme with catalase- and superoxide dismutase-mimetic activities, effectively scavenging ROS and inhibiting GSDMD-mediated pyroptosis. Compared with traditional anti-pyroptosis drugs, the CaMn nanozyme demonstrates significant advantages: (1) multifunctional synergistic effects: simultaneously scavenging ROS and inhibiting pyroptosis, whereas existing drugs (e.g., pyroptosis inhibitors) typically target single pathways; (2) superior material stability with sustained catalytic activity, overcoming the frequent dosing requirements of small-molecule antioxidants like disulfiram; and (3) a wider therapeutic window with more flexible dosing compared to drugs with narrow toxicity margins such as disulfiram.²⁷In vitro experiments confirmed that the constructed CaMn nanozyme significantly reduced pyroptotic cell death under inflammatory conditions. Furthermore, in a murine colitis model, the nanozyme alleviated intestinal inflammation, maintained epithelial barrier integrity, and downregulated pyroptosis-related markers. Our findings demonstrate that pancatalytic therapy targeting pyroptosis represents a promising strategy for IBD treatment by simultaneously modulating oxidative stress and inflammatory cell death. After oral administration, nanozymes are mainly retained in the intestines, taken up by goblet cells and parietal cells, and some particles enter the systemic circulation through the lymphatic or portal system. In the liver and reticuloendothelial system, the surface of nanozymes is coated with protein coronas and engulfed by macrophages, and then gradually degraded in lysosomes, and the released metal ions (such as Mn²⁺) are excreted through the kidneys or bile.²⁸ It is worth noting that the enterohepatic circulation and potential neurotoxicity of manganese need to be further elucidated by isotope tracing and long-term toxicological studies.²⁹The non-specific action of the nanozyme may limit its efficacy in tissues with high baseline levels of ROS, highlighting the need for targeted delivery systems. Moreover, dose dependence remains a critical factor, with further research required to explore the therapeutic window and potential side effects at higher doses. Future research should focus on promoting clinical transformation of multi-dimensional optimization strategies. At the therapeutic level, it is necessary to explore its synergistic effects with biological agents (such as anti-TNF-α antibodies) to construct a multi-pathway combined treatment plan of “oxidative stress-inflammation-pyroptosis”.³⁰ In terms of delivery systems, colon-specific intelligent delivery technologies should be developed, including inflammation targeting and bacterial enzymatic substrate modification layers to enhance targeting and reduce systemic exposure risks.³¹ As for safety assessment, it is necessary to clarify the enterohepatic circulation of manganese through isotope labeling technology, focus on evaluating the blood–brain barrier penetration potential and neurotoxicity under long-term administration, and establish population-specific dose safety thresholds.²⁹ In addition, the size–surface charge–crystal phase parameters of nanozymes can be optimized in combination with organoid models and AI prediction tools to dynamically adapt them to the microenvironmental characteristics of different pathological stages of IBD (acute phase/mucosal healing phase), ultimately achieving a therapeutic upgrade from “broad-spectrum antioxidant” to “precise dynamic regulation”.^32,33 This innovative therapeutic approach offers a novel “kill two birds with one stone” strategy for IBD and related inflammatory disorders. Its potential to overcome the limitations of current therapies has been preliminarily validated. However, successful clinical translation will ultimately depend on precision engineering of a drug delivery system and synergistic development of combination therapies.

Author contributions

Li Ding, Meiqi Chang and Yu Chen conceived and designed this project. Lili Liu, Wenting Ye and Yanqiu Duan performed the experiments. Meiqi Chang, Lili Liu, Wenting Ye and Xinran Song analyzed the data. Meiqi Chang, Lili Liu and Li Ding wrote the paper. Yu Chen revised the paper. All authors discussed the experimental procedures and results.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5tb01210d

Acknowledgements

This work was financially supported by Shanghai Science and Technology Committee Rising-Star Program (24QA2708600), Shanghai Young Talent Program of Eastern Talent Plan (QNWS2024006), and Wenzhou Basic Scientific Research Project (Grant No. Y20240112, Y2023136), the National Key Research and Development Projects (Grants No. 2023YFC2306500).

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Footnote

† Lili Liu and Wenting Ye contributed equally to this work.

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