Biological bionic hybrid membrane coated prussian blue nano-enzyme as well as preparation method and application of biological bionic hybrid membrane coated prussian blue nano-enzyme

Abstract

The invention discloses a biological bionic hybrid membrane coated Prussian blue nano-enzyme, which comprises a Prussian blue nano-enzyme and a neutral granulocyte membrane (N)-macrophage membrane (M) hybrid membrane (NM hybrid membrane), and the Prussian blue (PB) nano-enzyme is coated in the NM hybrid membrane. Compared with an N-PB nano-enzyme and an M-PB nano-enzyme, the hybrid membrane NM-PB nano-enzyme is remarkably improved in the aspects of targeting force and ulcerative colitis treatment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Prussian blue nanozyme wrapped in a biomimetic hybrid membrane, a preparation method and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic and relapsing disease that affects the gastrointestinal tract and belongs to the category of inflammatory bowel disease (IBD). At present, the main drugs for the clinical treatment of UC are aminosalicylic acid, antibiotics, corticosteroids and immunosuppressants. However, the efficacy of these treatments is often poor and is often accompanied by significant adverse reactions. The etiology and pathogenesis of IBD are related to multiple factors, including impaired intestinal epithelial barrier function, immune dysregulation and intestinal flora dysbiosis. The presence of UC can lead to intestinal immune dysregulation, and immune cells infiltrate into inflamed colon tissue, resulting in excessive production of reactive oxygen species (ROS), which triggers cellular oxidative stress damage and exacerbates the inflammatory response. Traditional antioxidants face challenges in targeted delivery and often fail to effectively reach the lesion site. Therefore, innovative therapeutic strategies are urgently needed to treat UC.

[0003] ROS scavenging therapy aims to remove ROS by targeted delivery of antioxidant materials into inflamed tissues with an unbalanced redox environment. Nanozymes have the ability to scavenge ROS and have become an alternative to traditional enzymes and have been widely used in the treatment of tumors and ROS-related diseases. Prussian blue (PB) nanozymes have been approved for marketing by the US Food and Drug Administration (FDA) and have the characteristics of hemostatic stability, biocompatibility, and low cytotoxicity. They can effectively eliminate ROS, including ·OH, H2O2, and ·OOH. However, nanodrug delivery faces multiple biological barriers, including interaction with blood, limited tissue penetration, and inefficient cellular uptake. In addition, the underlying molecular mechanism of PB nanozymes in the treatment of UC remains unclear. Therefore, it is crucial to develop a safe and effective treatment strategy to modify PB nanozymes for targeted delivery to UC lesions. Summary of the invention

[0004] The object of the present invention is to provide a biomimetic hybrid membrane-wrapped Prussian blue (PB) nanozyme (also referred to herein as "NM-PB nanozyme"). After being wrapped by the NM hybrid membrane, the PB nanozyme specifically targets the ulcerative colitis (UC) lesion site, thereby enhancing the delivery of the PB nanozyme targeted to UC and enhancing the efficacy of the PB nanozyme in treating UC.

[0005] To achieve the purpose of the present invention, the following embodiments are provided.

[0006] In one embodiment, a biomimetic hybrid membrane-encapsulated Prussian blue nanozyme of the present invention comprises a Prussian blue nanozyme (PB) and a hybrid membrane (NM hybrid membrane) of a neutrophil membrane (N)-macrophage membrane (M), and the Prussian blue (PB) nanozyme is encapsulated in the NM hybrid membrane.

[0007] In some embodiments, in the above-mentioned Prussian blue nanozyme of the present invention, the protein mass ratio of neutrophil membrane to macrophage membrane is 1:1.

[0008] In some embodiments, in the above-mentioned Prussian blue nanozyme of the present invention, the mass ratio of the NM hybrid membrane to the Prussian blue nanozyme is 1:1.

[0009] Another object of the present invention is to provide a method for preparing the Prussian blue nanozyme wrapped by the biomimetic hybrid membrane of the present invention, comprising the following steps:

[0010] 1) Take neutrophils and macrophages, and use membrane protein extraction kit to extract neutrophil membrane (N) and macrophage membrane (M), respectively;

[0011] 2) Mixing the neutrophil membrane (N) and the macrophage membrane (M), and ultrasonically treating them to obtain a NM hybrid membrane;

[0012] 3) The NM hybrid membrane and the PB nanozyme are mixed and ultrasonically treated in an ice bath to obtain the Prussian blue nanozyme wrapped in the NM hybrid membrane.

[0013] In some embodiments, in the above-mentioned preparation method of the present invention, in step 2), the mass ratio of the neutrophil membrane (N) to the macrophage membrane (M) is 1:1.

[0014] In some embodiments, preferably, in the above-mentioned preparation method of the present invention, in step 2), the ultrasonic treatment refers to ultrasonic treatment at 37° C. for 10 minutes.

[0015] In some embodiments, preferably, in the preparation method of the present invention, in step 3), the ultrasonic treatment time is 10 minutes.

[0016] In another embodiment, the present invention also provides a use of the Prussian blue nanozyme wrapped by the biomimetic hybrid membrane of the present invention in the manufacture of a drug for treating ulcerative colitis.

[0017] Technical effects:

[0018] The Prussian blue nanozyme (abbreviated as biomimetic NM-PB nanozyme) wrapped by the biomimetic hybrid membrane of the present invention shows significant targeting ability in both the cellular colitis model and the mouse ulcerative colitis (UC) model compared with N-PB and M-PB nanozymes, and significantly enhances the localization and uptake of PB nanozymes in the lesion site. The oxidative stress level in the affected (acting) area can be more effectively reduced, and the redox balance is effectively regulated. The excessive immune response of inflammatory cells is suppressed, and the level of proinflammatory cytokines is significantly reduced. In addition, the reduction of ROS further reduces the secondary damage of the intestine and significantly reduces the apoptosis of intestinal epithelial cells. In addition, NM-PB nanozymes effectively restore the barrier function of intestinal epithelial cells and promote the polarization of M1 macrophages to M2. Through mechanism research, it is found that NM-PB nanozymes effectively and significantly inhibit the progression of UC by inhibiting the signaling pathway related to cytokine-cytokine receptor interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Characterization of the characteristics of NM-PB nanozymes and their in vitro targeting ability, including A) Scanning electron microscopy (SEM, scale bar = 200 nm) and transmission electron microscopy (TEM, scale bar = 20 nm) images of PB, N-PB, M-PB, and NM-PB nanozymes. B) X-ray diffraction (XRD) patterns of PB nanozymes. C) X-ray photoelectron spectroscopy (XPS) analysis of PB nanozymes in the Fe 2p region. DE) Dynamic light scattering (DLS) and Zeta potential analysis of PB, N-PB, M-PB, and NM-PB nanozymes. F) Iron concentrations in each group measured by inductively coupled plasma mass spectrometry (ICP-OES) (n = 3). G) Confocal laser scanning microscopy (CLSM) images of neutrophil-macrophage hybrid membranes (scale bar = 10 μm); SDS-PAGE protein analysis of PB nanozymes, N, M, NM, N-PB nanozymes, M-PB nanozymes, and NM-PB nanozymes by Coomassie Brilliant Blue staining. H) Intracellular uptake of N-PB, M-PB and NM-PB nanozymes (blue represents cell nuclei stained with DAPI, red represents N-PB, M-PB and NM-PB nanozymes stained with Dil) (Scale bar = 20 μm) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0020] Figure 1S The figures show the biocompatibility of NM-PB nanozymes and the experimental results of studying their potential anti-inflammatory effects in vitro, where A) the survival rate of FHC cells after incubation with NM-PB nanozymes, and B) the survival rate of RAW264.7 cells after incubation with NM-PB nanozymes.

[0021] Figure 2 Figure 3 is the experimental results of the reactive oxygen species (ROS) scavenging activity of NM-PB nanozymes and their ability to induce macrophage reprogramming in vitro, wherein AC) the scavenging ability of NM-PB nanozymes to hydroxyl radicals (·OH), peroxyl radicals (·OOH) and hydrogen peroxide (H2O2); DE) the intracellular ROS scavenging ability of NM-PB nanozymes detected by flow cytometry and confocal laser scanning microscopy (CLSM, scale bar = 10 μm); F) real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR, n =3) Relative mRNA levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in FHC cells after different treatments; G) Immunofluorescence staining of TNF-α in FHC cells after different treatments (scale bar = 10 μm); HI) Immunofluorescence staining of iNOS and CD206 in RAW264.7 cells after different treatments (scale bar = 10 μm); J) Western blot analysis of E-cadherin, Occludin, Bcl2 and Bax protein expression in FHC cells after different treatments, *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001, one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0022] Figure 3 Figure 1: Experimental results for determining the optimal dose of Prussian blue (PB) nanozyme for treating UC model, where A) Schematic diagram of the experimental scheme for treating UC using PB nanozyme; B) Changes in mouse body weight after different treatments; C) Changes in disease activity index (DAI) after different treatments; D) Macroscopic appearance of colon tissue after different treatments and representative images of hematoxylin-eosin (H&E) staining (scale bar = 200 μm). E) Colon length (n = 5); FH) mRNA expression of TNF-α, IL-1β and IL-6 determined by RT-qPCR after different treatments (n = 4); I) Protein expression of Occludin, Bcl2 and Bax analyzed by Western blot after different treatments; *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001, using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0023] Figure 3SThe results of the in vivo biosafety assessment of NM-PB nanozymes are shown in Figure 1, including: AB) biochemical indicators of liver function, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST); CD) biochemical indicators of renal function, including blood urea nitrogen (BUN) and creatinine (CREA); EF) routine blood examination parameters, including red blood cell count and white blood cell count; G) hematoxylin-eosin (H&E) staining of major organs (heart, liver, spleen, lung and kidney) after different treatments.

[0024] Figure 4 The results of the in vivo targeting ability experiment of NM-PB nanozyme, A) in vitro fluorescence imaging of colon tissues of each group; B) quantitative analysis of the average fluorescence intensity of colon tissues (n = 4); C) in vitro fluorescence imaging of major organs; D) autofluorescence confocal laser scanning microscopy (CLSM) images of colon tissue sections (blue fluorescence indicates the presence of cell nuclei, while red fluorescence corresponds to N-PB, M-PB and NM-PB nanozymes labeled with Dil; scale bar = 500 microns); E) quantification of iron concentration measured by inductively coupled plasma mass spectrometry (ICP-OES) after different treatments (n = 3). *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001, using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0025] Figure 5 The figure shows the therapeutic effect of NM-PB nanozyme in the mouse UC model, where A) Schematic diagram of the experimental scheme for treating UC using PB, N-PB, M-PB, and NM-PB nanozymes; B) Changes in the body weight of mice after different treatments; C) Changes in the disease activity index (DAI) after different treatments; D) Macroscopic appearance of colon tissue and representative images of hematoxylin-eosin (H&E) staining (scale bar = 200 μm); E) Colon length (n = 6); FH) IL-6, TNF-α, and myeloperoxidase (MPO) levels measured in colon tissue by enzyme-linked immunosorbent assay (ELISA) (n = 5); IK) mRNA expression of TNF-α, IL-1β, and IL-6 measured in colon tissue by RT-qPCR after different treatments. L) Protein expression of Occludin and Bcl2 analyzed in colon tissue by Western blot after different treatments. *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001, one-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used.

[0026] Figure 6Figure 2 shows that NM-PB nanozymes restore mucosal barrier function and promote macrophage reprogramming in ulcerative colitis (UC) mice, including A) immunofluorescence staining of ZO-1 and Occludin in colon tissues after different treatments (scale bar = 200 μm); B) representative images of terminal deoxyribonucleotidyl transferase-mediated nick end labeling (TUNEL) staining in colon tissues after different treatments (scale bar = 200 μm); C) immunofluorescence staining of iNOS and CD206 expression in colon tissues after different treatments (scale bar = 200 μm).

[0027] Figure 7 Figure 3 RNA-seq analysis of NM-PB nanozyme regulating DSS-induced colitis, where A) Venn diagram shows the differentially expressed genes (DEGs) identified in the PBS group, DSS group, PB group and NM-PB group by whole transcriptome RNA-seq analysis (|log2(fold change)|>1, P<0.05 after correction); B) Gene Ontology (GO) functional enrichment analysis of the NM-PB group and the DSS group; C) Volcano plot shows the differentially expressed genes (up-regulated genes: red; down-regulated genes: blue) between the DSS group and the PBS group (left), the PB group and the DSS group (middle), and the NM-PB group and the DSS group (right) in the RNA-seq data.

[0028] Figure 7S Figure 1. Volcano plot and bar graph of total differentially expressed genes (DEGs), including A) principal component analysis (PCA) revealed the differences in transcriptome profiles among the PBS, DSS, PB and NM-PB groups, B) heat map generated by RNA-seq analysis revealed the unique transcriptome features among the PBS, DSS, PB and NM-PB groups, C) bar graph of differentially expressed genes (DEGs) showed the expression levels between the DSS and PBS groups, the NM-PB and DSS groups, the NM-PB and PBS groups, and the PB and DSS groups, D-F) based on the transcriptome differentially expressed genes (DEGs) data, the expression changes of Cxcl3, Il19 and Il20ra in colon tissues after different treatments were verified.

[0029] Figure 8The therapeutic mechanism of NM-PB nanozyme in the mouse UC model, including: A-C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) between DSS group and PBS group, PB group and DSS group, and NM-PB group and DSS group; D-G) mRNA expression of Csf3, Cxcl2, Cxcl3 and Il20ra determined by RT-qPCR in colon tissues after different treatments. H) Expression verification of Csf3, Cxcl2, Cxcl3 and Il20ra in colon tissues after different treatments measured by RT-qPCR and Western blot, *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used. DETAILED DESCRIPTION

[0030] The following examples are representative and are used to further understand and illustrate the spirit of the present invention, but are not intended to limit the scope of the present invention in any way.

[0031] The sources of reagents used in the following examples are:

[0032] The reagents used for the synthesis of Prussian blue (PB), namely, citric acid, FeCl3·6H2O, and potassium ferrocyanide K4[Fe(CN)6]·3H2O, were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

[0033] 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil), 3,3′-dioctadecyloxycarbocyanine iodide (Dio), and 4′,6-diamidino-2-phenylindole (DAPI) were provided by Biotechnology Biotech Co., Ltd. (Shanghai, China).

[0034] The compound 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindoltricarbocyanine iodide (Dir) was purchased from MedChemexpress Biotechnology (New Jersey, USA).

[0035] Lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (MO, USA), and dextran sulfate sodium (DSS) was purchased from Wuhan Core Biotechnology Co., Ltd. (Wuhan, China).

[0036] β-actin (ab8227, Abcam), ZO-1 (1:1000 dilution, #5406, Cell Signaling Technology), Occludin polyclonal antibody (1:1000 dilution, 27260-1-AP, Proteintech), E-cadherin (1:1000 dilution, 20874-1-AP, Proteintech), Bcl2 monoclonal antibody (1:1000 dilution, ab182858, Abcam), and Bax monoclonal antibody (1:1000 dilution, ab32503, Abcam).

[0037] Horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse antibodies were used as secondary antibodies (1:5000 dilution, Cell Signaling Technology, Inc.). Dylight 549 goat anti-rabbit IgG was purchased from Abbkine, Wuhan, China.

[0038] Example 1 Synthesis of Prussian Blue Nanozyme

[0039] The synthesis of Prussian blue nanozyme follows the method described in the previous literature [Liang X, Deng Z, Jing L, LiX, Dai Z, Li C, Huang M. Prussian blue nanoparticles operate as a contrast agent for enhanced photoacoustic imaging. Chem Commun (Camb). 2013; 49: 11029-1103131, referenced]. The preparation steps are as follows:

[0040] 1. Dissolve 1.2 g of citric acid and 135.15 mg of FeCl3·6H2O in 500 mL of double distilled water to prepare solution 1;

[0041] 2. Dissolve 1.2 g of citric acid and 211.2 mg of potassium ferrocyanide K4[Fe(CN)6]·3H2O in 500 mL of double distilled water to prepare solution 2;

[0042] 3. Continuously stir solution 2 with magnetic stirring at 60°C, and gradually add solution 1 dropwise. The mixed solution turns into transparent blue, and continue stirring until it reaches room temperature.

[0043] 4. Centrifuge the solution in the previous step (500,000×g, 60 minutes) to separate the Prussian blue nanozyme (PB-nanozyme), and store it at 4°C for future use.

[0044] PB-nanozyme characterization:

[0045] The morphology and size of PB nanozymes can be clearly observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. Figure 1 A). The results showed that PB nanozyme exhibited a clear cubic crystal structure. The X-ray diffraction (XRD) pattern is shown in Figure 1 As shown in B, obvious diffraction planes of PB are displayed at 17.5° (200), 24.8° (220), 35.4° (400) and 39.7° (420).

[0046] The chemical state of PB was characterized using X-ray photoelectron spectroscopy (XPS). Figure 1 C, in Fe2P 3 / 2 (711.98 eV) and Fe2P 1 / 2 The peak observed at (720.78 eV) corresponds to the Fe III The peak at 707.88 eV indicates the presence of Fe3[Fe2(CN)6] 4- Fe2P 3 / 2 .

[0047] Example 2 Preparation of biomimetic NM-PB nanozyme

[0048] Venous blood samples were collected from a group of healthy human volunteers. The research in the present invention has been approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (approval number: 61 / 2024). In addition, periorbital blood was collected from C57BL / 6 mice. All animal experiments involved in the present invention have been approved by the Experimental Animal Care and Use Committee of Chongqing Medical University (IACUC-COMU) (approval number: IACUC-COMU-2024-0551).

[0049] Neutrophils from human samples were isolated using human neutrophil isolation medium (TBD, LZS11131), and neutrophils from mouse samples were isolated using mouse peripheral blood neutrophil isolation kit (TBD, LZS100).

[0050] Human and mouse macrophage membranes were obtained from PMA-activated THP-1 cells and RAW264.7 cells, respectively.

[0051] Neutrophil membrane (N) and macrophage membrane (M) were extracted from neutrophils and macrophages, respectively, using a membrane protein extraction kit (Beyotime, P0033).

[0052] Neutrophil membranes (N) and macrophage membranes (M) were mixed at a protein weight ratio of 1:1 and sonicated at 37°C for 10 minutes to obtain neutrophil-macrophage hybrid membranes (NM hybrid membranes).

[0053] The human NM hybrid membrane (200 μg / mL) was mixed with an equal volume of PB nanozyme at a concentration of 200 μg / mL and sonicated in an ice bath for 10 min to obtain NM-PB nanozyme by sonication.

[0054] Neutrophil membrane (N) (200 μg / mL) and macrophage membrane (M) (200 μg / mL) were mixed with equal volumes of PB nanozyme at a concentration of 200 μg / mL, and ultrasonically treated in an ice bath for 10 min to form N-PB nanozyme and M-PB nanozyme (as subsequent comparative experimental samples) by ultrasonic method.

[0055] The mouse NM-PB nanozymes formed by the neutrophil membrane (N) (200 μg / mL) and macrophage membrane (M) of the above mice were used as animal modeling.

[0056] Characterization of NM-PB nanozymes:

[0057] The cell membrane fragments and cell membrane-encapsulated PB nanozymes were characterized using confocal laser scanning microscopy (CLSM) (Nikon, Japan), transmission electron microscopy (TEM) (JEOL JEM-2100 Plus, Japan), scanning electron microscopy (SEM) (Zeiss GeminiSEM 300, Germany), and Coomassie brilliant blue staining. The particle size and surface charge of the nanoparticles were evaluated using a nanoparticle size and zeta potential analyzer (Shanghai Optech Technology Co., Ltd.).

[0058] For visualization, the neutrophil membrane (N) was labeled with Dio (200 μg / mL) and the macrophage membrane (M) was labeled with Dil.

[0059] Dio-labeled N and Dil-labeled M were mixed at a protein weight ratio of 1:1 and sonicated at 37°C for 10 min to obtain a Dio- and Dil-labeled NM hybrid membrane.

[0060] The Dio- and Dil-labeled NM hybrid membrane (200 μg / mL) was mixed with an equal volume of PB nanozyme at a concentration of 200 μg / mL and sonicated in an ice bath for 10 min to obtain Dio- and Dil-labeled NM-PB nanozyme by sonication for subsequent visualization studies and characterization.

[0061] NM hybrid membrane characterization:

[0062] Scanning microscopy (CLSM) showed that the fluorescence signals from the NM hybridization membranes were significantly colocalized ( Figure 1 G).

[0063] Coomassie Brilliant Blue staining analysis showed that the protein bands observed on the NM-PB nanozyme were comparable to those of the separated N and M components, as indicated by the green and red arrows, thus indicating the presence of membrane proteins ( Figure 1 G) Through direct observation by TEM and SEM, it was confirmed that there was a thin film coating on the surface of NM-PB, N-PB and M-PB nanozymes. Figure 1 As shown in DE.

[0064] Dynamic light scattering (DLS) analysis showed that the average particle size of PB was 108.3 nm. After modification with cell membrane, the size of nanozymes increased significantly: 156.5 nm for N-PB nanozymes, 154.1 nm for M-PB nanozymes, and 159.0 nm for NM-PB nanozymes.

[0065] Zeta potential measurements showed that NM-PB nanozymes (-19.0 mV), N-PB nanozymes (-28.4 mV), and M-PB nanozymes (-23.7 mV) exhibited significantly higher negative charges compared to unmodified PB (-17.1 mV). The presence of a strong negative Zeta potential on the cell membrane surface was responsible for this phenomenon, indicating that the PB nanozymes had been successfully modified by the cell membrane, i.e., encapsulated by N, M, and NM membranes.

[0066] Example 3 Targeting, biocompatibility and bioactivity experiments

[0067] 1. In order to evaluate the biological activity, targeting ability and in vivo efficacy of the NM-PB nanozyme of the present invention, the present invention provides the following experiments.

[0068] 1.1 Cell culture and cell activity assessment

[0069] Human fetal colon (FHC) cells and RAW264.7 cells were seeded in 96-well plates at a density of 7×103 cells per well. After culturing in Dulbecco's modified Eagle's medium (DMEM, Gibco, 11965) containing 10% fetal bovine serum (FBS, Procell, 164210) for 24 hours, the cells were treated with different concentrations of NM-PB nanozymes for 24 hours. Subsequently, the cell activity was evaluated using the CCK-8 kit (TargetMol, C0005).

[0070] 1.2 In vitro targeting ability of NM-PB nanozymes

[0071] Human fetal colon (FHC) cells were purchased from the American Type Culture Collection (#CRL-1831, ATCC, Manassas, VA, USA). FHC cells were seeded in confocal culture dishes at a density of 5×103 cells per well and cultured for 12 hours. To establish a colitis cell model, cells were treated with lipopolysaccharide (LPS) at a concentration of 100 μg / mL for 2 hours [Establishment of alipopolysaccharide induced inflammation model of human fetal colon cells. Mol Biol Rep. 2023; 50: 5557-5564. Reference]. Subsequently, Dil-labeled N-PB, M-PB and NM-PB nanozymes (PB 200 μg / mL) were added to LPS-induced FHC cells, respectively. After incubation for 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 minutes, and counterstained with DAPI for 10 minutes. Finally, the culture dish was washed three times with PBS, and the cellular uptake was detected by observing the fluorescence signal by CLSM.

[0072] 1.3 In vitro ROS scavenging effect of NM-PB nanozymes

[0073] Titanium dioxide (TiO2) produces hydroxyl radicals (·OH) under 340 nm UV light. NM-PB nanozymes (PB concentration of 200 μg / mL) were added to TiO2 (0.1 mg / mL) and 50 mM BMPO (a cyclic nitrone spin trap) and analyzed using an electron spin resonance (ESR) spectrometer. Xanthine (1 mM) and xanthine oxidase (0.2 U / mL) react to produce superoxide anions (·OOH). BMPO undergoes a spin addition reaction with ·OOH to form BMPO / ·OOH adducts. NM-PB nanozymes (PB concentration of 200 μg / mL) were added to the xanthine / xanthine oxidase system and detected using an ESR spectrometer. In addition, light (500 W bulb) can catalyze the generation of hole electrons from a hydrogen peroxide (H2O2) solution (1 mM). NM-PB nanozyme (equivalent PB concentration: 1 mg / mL) was added to the system, along with H2O2 solution (1 mM) and 2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO, 100 mM) system, and the resulting mixture was analyzed using an ESR spectrometer.

[0074] 1.4 In vivo ROS scavenging ability of NM-PB nanozymes

[0075] FHC cells were cultured in confocal culture dishes to establish a colitis cell model, and then incubated with PB, N-PB, M-PB, and NM-PB nanozymes (equivalent to a PB concentration of 200 μg / mL) for 12 hours. DCFH-DA (Biyuntian, S0033), a fluorescent dye commonly used for ROS detection, was then used to assess intracellular ROS levels. ROS levels in FHC cells were analyzed and quantified using confocal microscopy and flow cytometry.

[0076] 1.5 RT-qPCR and primers

[0077] Total RNA was extracted from FHC cells and mouse colon tissue using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Total RNA was reverse transcribed using the Takara reverse transcription kit. Real-time quantitative PCR (RT-qPCR) was performed using PremixEx TaqTM II (Takara #RR820A). The relative amounts were normalized to β-actin to determine the amount of β-actin. The primer sequences are listed in Table 1.

[0078] Table 1. Quantitative PCR primer sequences

[0079]

[0080] 1.6 Western-Blot

[0081] Total protein was extracted from FHC cells and mouse colonic lesion tissue using RIPA lysis buffer (P0013B, Beyotime Biotech Co., Ltd., China). Protein concentration was determined using the BCA protein assay (P0009, Beyotime Biotech Co., Ltd., China). Equal amounts of protein samples (30 μg) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck KGaA; Darmstadt, Germany). The PVDF membranes were blocked with 5% skim milk at 37 °C for 2 h and then incubated with primary antibodies at 4 °C overnight. Afterwards, the PVDF membranes were incubated with the corresponding secondary antibodies at room temperature for 2 h. Finally, the proteins on the membrane were visualized using an enhanced chemiluminescence (ECL) detection kit (EMD Millipore, Burlington, MA, USA) and evaluated using an ECL Western Blotting System (Bio-Rad Laboratories Inc., Hercules, CA, USA).

[0082] 1.7 Immunofluorescence analysis

[0083] After permeabilization, the samples were blocked with bovine serum albumin (BSA) for 2 h and then incubated with primary antibodies (1:200 dilution) at 4°C overnight. The next day, the samples were incubated with fluorescent secondary antibodies (1:500 dilution) at room temperature for 1 h. Afterwards, the samples were stained with DAPI and observed using a confocal laser scanning microscope (Nikon, Japan).

[0084] 1.8 Determining the optimal dose of PB nanozymes for the treatment of ulcerative colitis (UC)

[0085] C57BL / 6 mice (male, 6 weeks old, 17-18 g) were purchased from the Experimental Animal Center of Chongqing Medical University, China. To determine the optimal dose of PB nanozymes for the treatment of UC, the mice were randomly divided into five groups (n=5): 1) PBS group (healthy mice received PBS injection), 2) DSS group (DSS-induced colitis mice that did not receive treatment), 3) DSS+PB 8 mg / kg group (DSS-induced colitis mice that received 8 mg / kg PB nanozymes), 4) DSS+PB 20 mg / kg group (DSS-induced colitis mice that received 20 mg / kg PB nanozymes), and 5) DSS+PB 50 mg / kg group (DSS-induced colitis mice that received 50 mg / kg PB nanozymes). DSS was dissolved in double distilled water to prepare a 3% (w / v) solution, which was then orally administered to groups 2-5 for five consecutive days. On days 5, 6, and 7, PB nanozymes were intravenously injected into groups 3, 4, and 5 at corresponding doses. During this period, the body weight, stool consistency, and blood in the stool were assessed and recorded daily. The mice were euthanized on day 8, and colon tissues were immediately collected for histological analysis.

[0086] 1.9 In vivo targeting ability of NM-PB nanozymes

[0087] To evaluate the in vivo targeting ability of NM-PB nanozymes in ulcerative colitis (UC) lesions, Dir-labeled N-PB, M-PB, and NM-PB nanozymes were intravenously injected into DSS-induced UC mice. 24 hours after injection, ex vivo fluorescence imaging of the colon and major organs (heart, liver, spleen, lung, and kidney) was performed using the AniView in vivo imaging system (BLT Biotechnology; Guangzhou, China). The UC model mice were set as the DSS group, and the mean fluorescence intensity of the colon was calculated. In addition, these nanozymes were labeled with Dil instead of Dir fluorescent dye so that their distribution in UC lesions could be observed under a fluorescence microscope.

[0088] 1.10 In vivo therapeutic effect of NM-PB nanozyme

[0089] C57BL / 6 mice were randomly divided into six groups (n=6) as follows: 1) PBS group (healthy mice received PBS injection), 2) DSS group (untreated DSS-induced colitis mice), 3) DSS+PB group (DSS-induced colitis mice treated with PB nanozymes), 4) DSS+N-PB group (DSS-induced colitis mice treated with N-PB nanozymes), 5) DSS+M-PB group (DSS-induced colitis mice treated with M-PB nanozymes), and 6) DSS+NM-PB group (DSS-induced colitis mice treated with NM-PB nanozymes). In order to establish the UC model, mice in groups 2-6 were fed a feed containing 3% DSS for five consecutive days. On days 5, 6, and 7, mice in groups 3-6 received intravenous injections of PB, N-PB, M-PB, and NM-PB nanozymes, respectively. During the treatment period, the body weight, stool consistency, and blood in the stool of all mice were evaluated and recorded every day. On day 8, mice were euthanized and major organs (heart, liver, spleen, lung, kidney) and colon tissues were collected for histological analysis. In addition, blood samples were collected for serum biochemical tests and routine blood test.

[0090] 1.11 Enzyme-linked immunosorbent assay (ELISA)

[0091] The levels of proinflammatory cytokines and peroxidase in mouse colon tissues were quantified using ELISA kits for mouse IL-6, TNF-α, and MPO (purchased from Quanzhou Jiubang Biotechnology Co., Ltd., Fujian, China).

[0092] 1.12 The apoptosis of colon tissue cells was evaluated using the terminal deoxyribonucleotidyl transferase-mediated nick end labeling (TUNEL) detection kit from Roche (Mannheim, Germany). Apoptotic cells were observed using a fluorescence microscope (Olympus, Japan).

[0093] 1.13 RNA-seq analysis

[0094] After euthanasia of C57BL / 6 mice, colon cryosections were obtained for transcriptome analysis. Transcriptome sequencing includes multiple experimental steps, such as RNA extraction, sample quality control, library preparation, and sequencing on a sequencing platform. For bioinformatics analysis, the fragments per kilobase of transcript per million mapped reads (FPKM) were used to quantify the expression level of each transcript. Data analysis was performed using R software, and the raw counts were normalized using DESeq2. Differentially expressed genes (DEGs) were identified with a threshold of |log2(fold change)|≥1 and an adjusted P value (padj)<0.05 to generate volcano plots and principal component analysis (PCA) plots. In addition, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis were performed on the identified DEGs using Cluster Profiler. In addition, the expression changes of key genes in these pathways were validated by protein and RNA analysis in colon tissues.

[0095] 1.14 Statistical analysis

[0096] Statistical analysis was performed using GraphPad Prism software (version 7.03, GraphPad Software). Data are presented as mean ± SD. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by the Tukey-Kramer test (p < 0.05 was considered statistically significant, p < 0.01, p

[0097] <0.001, ****p<0.0001).

[0098] 2. Experimental results and analysis

[0099] 2.1 In vitro biocompatibility and targeting ability of NM-PB nanozymes

[0100] The non-toxicity of nanomedicines is an important prerequisite for their successful clinical transformation. In order to evaluate the biocompatibility of NM-PB nanozymes and study their potential anti-inflammatory effects in vitro, we performed cytotoxicity tests using FHC cells and RAW 264.7 immune cells. After FHC cells and RAW 264.7 cells were exposed to different concentrations of NM-PB nanozymes for 24 hours, the cell survival rate remained above 90% even at a high concentration of PB nanozymes (200 μg / mL), as shown in Figure 2. Figure 1S In AB, Figure 1SA) is the survival rate of FHC cells after incubation with NM-PB nanozymes, and B) is the survival rate of RAW264.7 cells after incubation with NM-PB nanozymes. These results confirm that NM-PB nanozymes have excellent biocompatibility, making them viable candidates for drug delivery applications in nanotherapeutics.

[0101] To further investigate the targeting ability of NM-PB nanozymes, we established a colitis cell model.

[0102] [Establishment of a lipopolysaccharide induced inflammation model of human fetal colon cells. Mol Biol Rep. 2023; 50: 5557-5564. References included]. The targeting efficiency of NM-PB nanozymes to FHC cells was subsequently evaluated. The cell membrane was labeled with Dil fluorescent dye for confocal laser scanning microscopy (CLSM) visualization. The cells were incubated with N-PB / Dil, M-PB / Dil, and NM-PB / Dil nanozymes (PB concentration of 200 μg / mL) for different times (2 hours, 4 hours, 8 hours, 12 hours, and 24 hours). CLSM imaging showed that the intracellular nanoparticle accumulation in the NM-PB group was significantly greater than that in the N-PB and M-PB groups ( Figure 1 H). To further verify this finding, we used inductively coupled plasma optical emission spectroscopy (ICP-OES) to measure the intracellular iron content. Compared with the PB group, N-PB group, and M-PB group, the NM-PB group had the highest iron content in the colitis cell model. This further suggests that the NM-PB nanozyme enhanced the uptake of the PB nanozyme in the colitis cell model ( Figure 1 F). Apparently, nanozymes modified with two cell membranes possess dual targeting capabilities, thus enhancing their enrichment and uptake at the lesion site.

[0103] 2.2 Evaluation of the ability of NM-PB nanozymes to scavenge reactive oxygen species (ROS) in vitro

[0104] Electron spin resonance (ESR) technology is widely used for the precise detection of free radicals due to its extremely high sensitivity. Therefore, we used ESR technology to measure the content of inflammation-related substances such as ·OH, ·OOH, and H2O2 after administration of NM-PB nanozymes. ESR results showed that the characteristic peak intensities of BMPO / ·OH, BMPO / ·OOH, and TEMPO / H2O2 were significantly reduced after the application of NM-PB nanozymes ( Figure 2 AC).

[0105] Excessive production of reactive oxygen species (ROS) caused by a comprehensive oxidative stress state in cells triggers a cascade of protein kinase reactions in inflammatory tissues, thereby promoting oxidative damage. The powerful antioxidant properties of NM-PB nanozymes prompted us to study its ability to restore cell survival under high oxidative stress conditions and evaluate the ability of NM-PB nanozymes to scavenge ROS in vitro. The LPS group was a colitis cell model established by inducing FHC cells with LPS, and then treated with equal doses of PB, N-PB, M-PB and NM-PB nanozymes, respectively. Subsequently, DCFH-DA staining was used to evaluate the level of ROS. Figure 2 As shown in E, the green fluorescence signal of FHC cells in the LPS group increased significantly. It is worth noting that compared with the LPS group, ROS levels were reduced after treatment with PB, N-PB, M-PB, and NM-PB nanozymes, among which NM-PB nanozymes showed the strongest ROS scavenging ability among all tested preparations. We further used flow cytometry to quantitatively analyze the intracellular ROS levels, and the results were consistent with the previous results ( Figure 2 D). These findings suggest that PB nanozymes with antioxidant enzyme activity can effectively scavenge ROS and protect cells from oxidative stress damage. Importantly, the antioxidant effect was particularly significant in the NM-PB group, suggesting that the dual-targeting ability of the neutrophil-macrophage hybrid membrane enhanced the cellular uptake of PB nanozymes, thereby improving their antioxidant effect.

[0106] Colonic epithelial cells in ulcerative colitis (UC) lesions are damaged by ROS, resulting in the release of a large number of damage-associated molecular patterns (DAMPs) into the intercellular space. These DAMPs stimulate macrophages to differentiate into a proinflammatory phenotype called M1 macrophages, which secrete various proinflammatory cytokines. These cytokines can further promote the production of intracellular ROS, thereby establishing a harmful cycle of inflammation and oxidative stress. The anti-inflammatory effect of NM-PB was confirmed by measuring inflammation-related cytokines through RT-qPCR. Figure 3 As shown in FH, the mRNA expression levels of IL-1β, TNF-α, and IL-6 were significantly reduced after treatment with PB, N-PB, M-PB, and NM-PB nanozymes compared with the LPS group. It is worth noting that NM-PB nanozymes showed particularly significant anti-inflammatory effects ( Figure 2 F). In addition, cell immunofluorescence analysis showed that the changes in TNF-α inflammatory factors in the colitis cell model were consistent with the above results. After NM-PB nanozyme treatment, the expression of TNF-α was significantly reduced ( Figure 2G). Together, these results suggest that NM-PB nanozymes have potent anti-inflammatory properties. Apoptosis of intestinal epithelial cells in UC can be attributed to secondary damage to intestinal cells by ROS. Apoptosis-related biomarkers were evaluated by Western Blot (WB), confirming the anti-apoptotic ability of NM-PB nanozymes. Figure 2 As shown in J, the expression of pro-apoptotic protein Bax was significantly inhibited in N-PB, M-PB and NM-PB groups compared with that in LPS group. In contrast, the expression of anti-apoptotic protein Bcl2 was upregulated ( Figure 2 J). It is noteworthy that among these groups, the NM-PB group showed stronger anti-apoptotic ability. Therefore, it can be inferred that NM-PB nanozymes effectively alleviated ROS-induced cell apoptosis.

[0107] Given the close correlation between intestinal barrier dysfunction and the pathogenesis of UC, as well as tight junction and adherens junction proteins (including ZO-1, occludin, and E-cadherin), we hypothesized that NM-PB nanozymes could restore impaired intestinal barrier function in the LPS-induced cellular colitis model. Subsequently, various treatments including PB, N-PB, M-PB, and NM-PB nanozymes were performed on the colitis cellular model.

[0108] WB analysis results showed that compared with the PB, N-PB and M-PB groups, the upregulation of E-cadherin and Occludin expression in the NM-PB group was the most significant ( Figure 2 J).

[0109] Activated inflammatory macrophages play a key role in the pathophysiology of enterocolitis. LPS-stimulated RAW264.7 macrophages undergo activation, which is characterized by upregulation of inducible nitric oxide synthase (iNOS), a hallmark of M1 macrophage polarization, leading to the production of a large amount of reactive nitrogen species (RNS), including nitric oxide (NO). These RNS disrupt mitochondrial oxidative phosphorylation, resulting in up to a 10-fold increase in ROS release. To further elucidate the potential of NM-PB nanozymes in promoting the polarization of M1 macrophages toward an M2 phenotype, we performed cell immunofluorescence analysis. Our results showed that PB, N-PB, M-PB, and NM-PB nanozymes significantly attenuated LPS-induced M1 macrophage activation and downregulated iNOS expression compared with the LPS group. In addition, these treatments upregulated the expression of CD206, a surface protein marker associated with M2 macrophages. In particular, NM-PB nanozymes showed the most significant effect in inducing this transition ( Figure 2 HI).

[0110] The above experimental results show that NM-PB nanozymes can break the harmful feedback loop established by ROS and excessive immune response in the colitis cell model, thereby alleviating the apoptosis of intestinal epithelial cells and restoring the intestinal mucosal barrier function. In addition, it can promote the transformation of M1 macrophages to M2 phenotype. This effect may be attributed to the dual targeting ability of neutrophil-macrophage hybrid membranes, which enhances the intracellular accumulation of PB nanozymes and further improves their functional efficiency.

[0111] 2.3 Evaluation of the therapeutic effect of PB nanozymes in UC

[0112] The powerful reactive oxygen species (ROS) scavenging activity of Prussian blue (PB) in vitro prompted us to further study its therapeutic effect in the dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model. All mice were randomly divided into five groups (n=5): G1: PBS group (healthy mice received PBS injection), G2: DSS group (ulcerative colitis mice), G3: DSS+PB 8mg / kg group, G4: DSS+PB 20mg / kg group, G5: DSS+PB 50mg / kg group. Mice received drinking water containing 3% DSS for 5 days to induce an acute ulcerative colitis model, and then PB nanozymes were injected through the tail vein on days 5, 6, and 7. The optimal therapeutic dose of PB nanozymes (8mg / kg, 20mg / kg, 50mg / kg) was determined by treating the ulcerative colitis mouse model with different concentrations of PB nanozymes ( Figure 3 A).

[0113] To evaluate the protective effect of PB nanozymes on ulcerative colitis lesions, we measured the body weight, colon length and disease activity index (DAI) of mice according to the methods described in the existing literature [Funakoshi T, et al. A novel nf-κb inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice. J Crohns Colitis. 2012; 6: 215-225.41, referenced] and performed histological examination of the distal colon. Figure 3 The results of BE showed that compared with the G2 group, mice treated with different doses of PB nanozymes showed significant improvements in body weight, intestinal length, and DAI. Hematoxylin-eosin (HE) staining results showed that the crypts of the intestinal tissue of mice in the G2 group were completely distorted, goblet cells were severely lost, and inflammatory cell infiltration was obvious. In contrast, these pathological changes in the G3, G4, and G5 groups were alleviated. Interestingly, the treatment effects of the G4 and G5 groups were comparable, and both significantly reduced inflammatory damage in mice.

[0114] Subsequently, we investigated the effect of PB nanozymes on the expression of inflammatory cytokines in a mouse model of ulcerative colitis. RT-qPCR analysis showed that the mRNA expression levels of IL-1β, TNF-α, and IL-6 in mice in groups G3, G4, and G5 were reduced compared with those in group G2 ( Figure 3 FH). Likewise, the effects of G4 and G5 groups were comparable, further supporting the anti-inflammatory role of PB nanozymes in alleviating DSS-induced ulcerative colitis.

[0115] To explore whether PB can restore the epithelial barrier in ulcerative colitis mice, we injected different concentrations of PB (8 mg / kg, 20 mg / kg, 50 mg / kg) into the colitis mouse model. Western blot (WB) results showed that compared with the G2 group, the expression levels of occludin in the G3, G4, and G5 groups were significantly upregulated ( Figure 3 I). In addition, the induction of ROS was associated with increased expression of the pro-apoptotic protein Bax, which promotes apoptosis of intestinal epithelial cells. After treatment with PB nanozymes, we observed a significant downregulation of Bax, while upregulation of the anti-apoptotic protein Bcl-2 ( Figure 3 I). Notably, the G4 and G5 groups showed comparable effects in alleviating intestinal epithelial cell apoptosis.

[0116] The above experimental results show that PB nanozymes have anti-inflammatory and anti-apoptotic effects and can restore the function of the intestinal epithelial barrier. Given that the efficacy observed in the G4 and G5 groups was comparable, we selected the 20 mg / kg dose as the optimal dose for subsequent in vivo experiments.

[0117] 2.4 Biocompatibility of NM-PB nanozymes

[0118] Before in vivo application, we evaluated the biosafety of NM-PB nanozymes by analyzing hematological indices and histopathological examination of vital organs. Figure 3S As shown in Figures AF, administration of NM-PB nanozymes did not result in any significant abnormalities in liver function, kidney function, or routine blood parameters. In addition, histological analysis using hematoxylin and eosin (H&E) staining showed that no significant pathological changes were observed in major organs (including heart, liver, spleen, lung, and kidney) after NM-PB nanozyme treatment ( Figure 3S (G). These findings validated the complete absorption and metabolism of NM-PB nanozymes after intravenous injection, demonstrating their good biosafety. This supports their potential for in vivo applications and provides a feasible pathway for therapeutic translation from animal experiments to clinical applications.

[0119] 2.5 In vivo targeting ability and therapeutic effect of NM-PB nanozymes

[0120] Ensuring that nanozymes are accurately delivered to ulcerative colitis (UC) lesions is crucial to ensuring therapeutic efficacy. Although we have previously demonstrated the in vitro targeting ability of NM-PB nanozymes, this study focused on evaluating its localization in UC lesions in a mouse model. For ease of observation, we labeled the cell membrane of the nanozyme with Dir fluorescent dye. After intravenous injection of equimolar amounts of N-PB, M-PB, and NM-PB nanozymes into the mouse UC model, colon tissue was collected 24 hours later for fluorescence imaging to observe its distribution. Figure 4 A and Figure 4 As shown in B, the fluorescence signals of the N-PB group and the M-PB group were weak, while a strong fluorescence signal was detected in the NM-PB group, indicating that the neutrophil-macrophage hybrid membrane has significant targeting ability. In addition, we also evaluated the biodistribution of the injected nanozymes in major organs. Biodistribution analysis showed that 24 hours after intravenous injection, there were fluorescent signals in the liver and kidneys ( Figure 4 C), suggesting that these organs are involved in the clearance of nanozymes from the systemic circulation.

[0121] Subsequently, we used confocal laser scanning microscopy (CLSM) to investigate the in vivo targeting ability of NM-PB nanozymes to UC lesions. The cell membrane was labeled with Dil fluorescent dye for CLSM observation. CLSM examination was performed on frozen sections of diseased intestinal tissue 24 h after intravenous injection. Only a small amount of red fluorescence signal was detected in the colon lesions of the N-PB and M-PB groups, while a significant enhancement of the signal was observed in the NM-PB group ( Figure 4 D), which is consistent with the ex vivo fluorescence imaging results.

[0122] We also further quantified the enrichment of nanozymes in intestinal lesions by analyzing iron content by inductively coupled plasma optical emission spectroscopy (ICP-OES). The NM-PB group had significantly higher iron concentrations in the lesioned intestinal tissues compared with the PB, N-PB, and M-PB groups ( Figure 4 E), further confirming the excellent targeting ability of NM-PB nanozymes to UC lesions. Taken together, these results indicate that neutrophil-macrophage hybrid membranes exhibit excellent targeting ability to UC lesions.

[0123] To further compare the therapeutic effects of PB, N-PB, M-PB, and NM-PB nanozymes in UC, all mice were randomly divided into six groups: G1 group (healthy mice received PBS injection), G2 group (UC mice), G3 group (DSS+PB), G4 group (DSS+N-PB), G5 group (DSS+M-PB), and G6 group (DSS+NM-PB). The therapeutic effect was evaluated by assessing the weight changes, disease activity index (DAI), colon length, expression levels of proinflammatory cytokines, and histological analysis of colon sections of mice in each group. Compared with the G2 group, the G3, G4, G5, and G6 groups had longer colon lengths, higher body weights, and lower DAI scores ( Figure 5 AE), among which the improvement in the G6 group was the most obvious. Colon tissue morphological analysis showed that the colon crypts in the G2 group were severely destroyed, immune cells were widely infiltrated, and colon epithelial cells were significantly damaged. In contrast, the G6 group showed almost normal histological microstructure ( Figure 5 D) These results indicate that G6 treatment significantly improved the symptoms and histomorphological features of the colitis mouse model.

[0124] Western blot (WB) and tissue immunofluorescence analysis showed that the expression of tight junction proteins ZO-1 and Occludin in the inflammatory lesions of group G6 was significantly upregulated compared with those of groups G3, G4, and G5 (see Figure 5 L, Figure 6 A). These results strongly demonstrate that NM-PB nanozyme treatment can enhance the expression of tight junction proteins, promote the assembly of tight junction complexes, and effectively restore intestinal barrier function.

[0125] In addition, TUNEL staining also showed apoptosis in colon tissue. It is worth noting that the red fluorescence intensity of TUNEL staining in the G6 group was significantly reduced compared with the G3, G4, and G5 groups ( Figure 6 B). Correspondingly, the expression level of Bcl2 protein in the G6 group was significantly increased after treatment, indicating that NM-PB nanozymes can effectively alleviate DSS-induced intestinal epithelial cell apoptosis ( Figure 5 L). In addition, the expression of inducible nitric oxide synthase (iNOS) was significantly downregulated, while the expression of CD206 was significantly upregulated in the G6 group compared with the G3, G4, and G5 groups.

[0126] The above experimental results show that NM-PB nanozymes can effectively promote the polarization of macrophages from M1 phenotype to M2 phenotype, thereby providing anti-oxidative stress protection for intestinal epithelial cells ( Figure 6C). The hybrid membrane-modified delivery system utilizes the inflammatory chemotactic properties of neutrophil and macrophage membranes to promote the accumulation of PB nanozymes in UC lesions. This targeted delivery method can effectively utilize the multiple functions of PB nanozymes, including anti-inflammatory effects, anti-apoptotic activity, restoration of the intestinal mucosal barrier, and promotion of polarization of M1 macrophages to M2 phenotypes.

[0127] 2.6 Transcriptome analysis of the in vivo therapeutic mechanism of NM-PB nanozymes

[0128] To elucidate the therapeutic mechanism of NM-PB nanozymes in vivo and reveal the molecular mechanism and signaling pathway of its effectiveness in treating ulcerative colitis (UC), we performed RNA sequencing on colon tissues of four experimental groups (PBS group, DSS group, DSS+PB group, and DSS+NM-PB group). The results of principal component analysis (PCA) showed that the four groups had different transcriptome characteristics (see Figure 7S In addition, from the Venn diagram and heat map ( Figure 7 A, Figure 7S Their unique transcriptome signatures can also be seen in (B).

[0129] The volcano plot and bar chart of total differentially expressed genes (DEGs) showed that there were 7008 DEGs in the DSS group compared with the PBS group, including 4096 upregulated genes and 2912 downregulated genes. After PB nanozyme treatment, a total of 6016 DEGs (2627 upregulated genes and 3389 downregulated genes) were identified. Notably, after NM-PB nanozyme treatment, a total of 7756 DEGs were observed, including 2959 upregulated genes and 4797 downregulated genes ( Figure 7 C, Figure 7S Middle C). Notably, the transcriptome profile of UC mice treated with NM-PB nanozymes was similar to that of the PBS group.

[0130] Subsequently, we performed cluster analysis and enrichment analysis on the DEGs to elucidate the therapeutic mechanism of NM-PB nanozymes. Based on the gene ontology (GO) database, the DEGs were preliminarily divided into three categories: molecular function (MF), biological process (BP), and cellular component (CC). There were significant differences (p < 0.05) between the DSS group and the NM-PB group in multiple biological pathways, including cytokine binding, oxidoreductase activity, signal receptor binding, and signal receptor regulatory activity ( Figure 7 B).

[0131] We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and used the log2FC expression level of DEGs as the evaluation criterion to study the related items. DEGs were associated with the cytokine-cytokine receptor interaction signaling pathway in KEGG between the DSS group and the PBS group, the PB group and the DSS group, and the NM-PB group and the DSS group ( Figure 8 AC). Therefore, we speculated whether NM-PB nanozymes regulate the progression of UC through the cytokine-cytokine receptor interaction pathway. Based on the analysis of DEGs data, the expression of key molecules (Cxcl3, Il19, Il20ra) related to the cytokine-cytokine receptor interaction pathway was inhibited ( Figure 7S DF). The results of RT-qPCR and WB experiments consistently showed that the expression levels of Cxcl2, Cxcl3, Il20ra, and Csf3 were significantly decreased after PB and NM-PB treatment compared with the DSS group, and the expression levels in the NM-PB group were close to those in the PBS group ( Figure 8 DH).

[0132] In summary, the NM-PB nanozyme of the present invention mainly alleviates the symptoms of the mouse UC model by inhibiting the cytokine-cytokine receptor interaction pathway. This action breaks the vicious cycle of inflammation and reactive oxygen species (ROS), leading to reduced apoptosis, reduced inflammation, and restoration of intestinal barrier function. The PB nanozyme wrapped by the neutrophil-macrophage hybrid membrane constructed by the present invention is intended to achieve targeted treatment of UC and elucidate its potential molecular mechanism. The hybrid membrane composed of neutrophils and macrophages gives the PB nanozyme dual targeting ability, significantly improving its accumulation in the UC lesion site. Compared with the PB nanozyme wrapped only by neutrophil or macrophage membranes, this design has obvious advantages.

[0133] NM-PB nanozymes showed strong ROS scavenging ability in both cellular colitis models and mouse ulcerative colitis models. They effectively inhibited the inflammatory response, reduced apoptosis of intestinal epithelial cells, restored the integrity of the intestinal mucosal barrier, and promoted the polarization of macrophages from M1 to M2. In addition, we found that NM-PB nanozymes can effectively slow down the progression of UC in mouse models by inhibiting the cytokine-cytokine receptor interaction signaling pathway. Importantly, NM-PB nanozymes showed excellent biocompatibility both in vivo and in vitro. Therefore, NM-PB nanozymes have great potential in clinical applications and provide a new direction for the application of antioxidant nanozymes in UC and other potential diseases.

Claims

1. A Prussian blue nanozyme wrapped in a biomimetic hybrid membrane, characterized in that: A hybrid membrane (NM hybrid membrane) comprising Prussian blue nanozyme and neutrophil membrane-macrophage membrane, wherein the Prussian blue (PB) nanozyme is encapsulated in the NM hybrid membrane.

2. The Prussian blue nanozyme as described in claim 1, wherein the protein mass ratio of neutrophil membrane to macrophage membrane is 1:

1.

3. The Prussian blue nanozyme according to claim 1, wherein the mass ratio of the neutrophil membrane-macrophage membrane hybrid membrane to the Prussian blue nanozyme is 1:

1.

4. A method for preparing the Prussian blue nanozyme wrapped by the biomimetic hybrid membrane of claims 1-3, comprising the following steps: 1) Take neutrophils and macrophages, and use membrane protein extraction kit to extract neutrophil membrane (N) and macrophage membrane (M), respectively; 2) Mixing the neutrophil membrane (N) and the macrophage membrane (M), and ultrasonically treating them to obtain a NM hybrid membrane; 3) The NM hybrid membrane and the PB nanozyme are mixed and ultrasonically treated in an ice bath to obtain the Prussian blue nanozyme wrapped in the NM hybrid membrane.

5. The preparation method according to claim 4, wherein in step 2), the mass ratio of the neutrophil membrane (N) to the macrophage membrane (M) is 1:

1.

6. The preparation method according to claim 4, wherein in step 2), the ultrasonic treatment refers to ultrasonic treatment at 37°C for 10 minutes.

7. The preparation method according to claim 4, wherein in step 3), the ultrasonic treatment time is 10 minutes.

8. Use of the Prussian blue nanozyme wrapped by the biomimetic hybrid membrane of claims 1-3 in the manufacture of a drug for treating ulcerative colitis.

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