A biomimetic hybrid membrane-wrapped Prussian blue nanozyme and its preparation method and application

Abstract

The present invention discloses a biomimetic hybrid membrane-encapsulated Prussian blue nanozyme, comprising a hybrid membrane (NM hybrid membrane) of a Prussian blue nanozyme and a neutrophil (N)-macrophage (M) membrane, wherein the Prussian blue (PB) nanozyme is encapsulated within the NM hybrid membrane. The hybrid membrane NM-PB nanozyme exhibits significant improvements in targeting and treatment of ulcerative colitis compared to N-PB and M-PB nanozymes.

Description

Technical Field

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

[0002] Ulcerative colitis (UC) is a chronic and relapsing disease that affects the gastrointestinal tract and falls under the category of inflammatory bowel disease (IBD). Currently, the main drugs used in 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 imbalance. The presence of UC can lead to intestinal immune dysregulation, with immune cells infiltrating into inflamed colonic tissue, resulting in excessive production of reactive oxygen species (ROS), which in turn 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 eliminate 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). They have the characteristics of hemostatic stability, biocompatibility and low cytotoxicity, and can effectively eliminate ROS, including ·OH, H2O2 and ·OOH. However, nano drug delivery faces multiple biological barriers, including interaction with blood, limited tissue penetration and low cellular uptake efficiency. In addition, the potential molecular mechanism of PB nanozymes in treating UC remains unclear. Therefore, it is crucial to develop a safe and effective treatment strategy to modify PB nanozymes to achieve targeted delivery to UC lesions. Summary of the Invention

[0004] The purpose of the present invention is to provide a biomimetic hybrid membrane-wrapped Prussian blue (PB) nanozyme (also referred to as "NM-PB nanozyme" herein). After being wrapped by the NM hybrid membrane, the PB nanozyme specifically targets the PB nanozyme to the ulcerative colitis (UC) lesion site, thereby enhancing the delivery of the PB nanozyme 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 Prussian blue nanozyme of the present invention, the protein mass ratio of the neutrophil membrane to the macrophage membrane is 1:1.

[0008] In some embodiments, in the 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 in the biomimetic hybrid membrane of the present invention, comprising the following steps:

[0010] 1) Neutrophils and macrophages were collected and membrane protein extraction kits were used to extract neutrophil membranes (N) and macrophage membranes (M).

[0011] 2) Mixing neutrophil membrane (N) and macrophage membrane (M) and sonicating to obtain NM hybrid membrane;

[0012] 3) The NM hybrid membrane and the PB nanozyme were 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 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 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 effect:

[0018] The Prussian blue nanozyme (abbreviated as biomimetic NM-PB nanozyme) wrapped in the biomimetic hybrid membrane of the present invention showed significant targeting ability in both the cellular colitis model and the mouse ulcerative colitis (UC) model compared to N-PB and M-PB nanozymes, significantly enhancing the localization and uptake of PB nanozymes at the lesion site. The oxidative stress level in the affected (action) area can be more effectively reduced, and the redox balance can be 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 to the intestine and significantly reduces the apoptosis of intestinal epithelial cells. In addition, NM-PB nanozyme effectively restores the barrier function of intestinal epithelial cells and promotes the polarization of M1 macrophages to M2. Through mechanism studies, it was found that NM-PB nanozyme effectively and significantly inhibits the progression of UC by inhibiting the signaling pathway related to cytokine-cytokine receptor interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 shows the characteristics of NM-PB nanozymes and their in vitro targeting ability. 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. D-E) Dynamic light scattering (DLS) and zeta potential analysis of PB, N-PB, M-PB, and NM-PB nanozymes. F) Iron concentration in each group determined by inductively coupled plasma mass spectrometry (ICP-OES) (n = 3). G) Confocal laser scanning microscopy (CLSM) image of neutrophil-macrophage hybrid membranes (scale bar = 10 μm); SDS-PAGE protein analysis of PB, N, M, NM, N-PB, M-PB, and NM-PB nanozymes stained with Coomassie Brilliant Blue. 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, among which A) the survival rate of FHC cells after incubation with NM-PB nanozymes, B) the survival rate of RAW264.7 cells after incubation with NM-PB nanozymes.

[0021] Figure 2 Figure 3 is an experimental result 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 on 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) the scavenging ability of NM-PB nanozymes detected by 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); H) 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: Results of an experiment to determine the optimal dose of Prussian blue (PB) nanozyme for treating UC models. A) Schematic diagram of the experimental protocol 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 and representative images of hematoxylin-eosin (H&E) staining of colon tissue after different treatments (scale bar = 200 μm). E) Colon length (n = 5); FH) TNF-α, IL-1β, and IL-6 mRNA expression by RT-qPCR after different treatments (n = 4); I) Western blot analysis of occludin, Bcl2, and Bax protein expression 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 3SFigure 1 shows the results of in vivo biosafety assessment of NM-PB nanozyme, 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 Figure 1 is a graph showing the in vivo targeting ability of NM-PB nanozymes, including: A) in vitro fluorescence imaging of colon tissues of each group; B) quantitative analysis of the mean 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 μm); E) quantification of iron concentration after different treatments by inductively coupled plasma mass spectrometry (ICP-OES) (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 Figure 2 shows the therapeutic effect of NM-PB nanozymes in a mouse UC model. A) Schematic diagram of the experimental protocol for treating UC using PB, N-PB, M-PB, and NM-PB nanozymes; 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 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) TNF-α, IL-1β, and IL-6 mRNA expression measured in colon tissue by RT-qPCR after different treatments. L) Occludin and Bcl2 protein expression 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 3: NM-PB nanozyme restores mucosal barrier function and promotes 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, including: A) Venn diagram showing 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, corrected P<0.05); B) Gene Ontology (GO) functional enrichment analysis of the NM-PB group and the DSS group; C) Volcano plot showing 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 5 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 8Figure 4: The therapeutic mechanism of NM-PB nanozymes in a mouse UC model. Figures A-C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) between the DSS and PBS groups, the PB and DSS groups, and the NM-PB and DSS groups. Figure 4: mRNA expression of Csf3, Cxcl2, Cxcl3, and Il20ra in colon tissues after different treatments, as measured by RT-qPCR. Figure 4: Expression of Csf3, Cxcl2, Cxcl3, and Il20ra in colon tissues after different treatments, as 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) with Tukey's multiple comparison test was 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 following examples use the following reagent sources:

[0032] The reagents used to synthesize 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 Beyotime Biotechnology Co., Ltd. (Shanghai, China).

[0034] The compound 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindole tricarbocyanine 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 Kerui 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-conjugated goat anti-rabbit and goat anti-mouse antibodies were used as secondary antibodies (1:5000 dilution, Cell Signaling Technology). Dylight 549 goat anti-rabbit IgG was purchased from Abbkine, Wuhan, China.

[0038] Example 1 Synthesis of Prussian Blue Nanozyme

[0039] Prussian blue nanozyme was synthesized following the method described previously [Liang X, Deng Z, Jing L, Li X, 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, incorporated by reference]. The preparation steps are as follows:

[0040] 1. Prepare solution 1 by dissolving 1.2 g of citric acid and 135.15 mg of FeCl3·6H2O in 500 mL of double-distilled water.

[0041] 2. Prepare solution 2 by dissolving 1.2 g of citric acid and 211.2 mg of potassium ferricyanide K4[Fe(CN)6]·3H2O in 500 mL of double-distilled water.

[0042] 3. Continuously stir solution 2 magnetically at 60°C while gradually adding solution 1 dropwise. The mixed solution turns transparent blue and continues 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 was as follows 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 As shown in 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 presence of Fe3[Fe2(CN)6] 4- Fe2P exists in 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 this 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 this invention have been approved by the Laboratory Animal Care and Use Committee of Chongqing Medical University (IACUC-COMU) (approval number: IACUC-COMU-2024-0551).

[0049] Neutrophils were isolated from human samples using Human Neutrophil Isolation Medium (TBD, LZS11131), and neutrophils were isolated from mouse samples 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 PB nanozyme at a concentration of 200 μg / mL in equal volumes 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 minutes 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 models.

[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 Optai Technology Co., Ltd.).

[0058] For visualization, neutrophil membranes (N) were labeled with Dio (200 μg / mL), and macrophage membranes (M) were 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] Dio- and Dil-labeled NM hybrid membrane (200 μg / mL) was mixed with 200 μg / mL PB nanozyme in equal volumes and sonicated in an ice bath for 10 minutes to obtain Dio- and Dil-labeled NM-PB nanozyme for subsequent visualization and characterization.

[0061] NM hybrid membrane characterization:

[0062] Scanning microscope (CLSM) showed that the fluorescence signals from the NM hybrid membrane 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, indicating the presence of membrane proteins ( Figure 1 G) Direct observation by TEM and SEM confirmed the presence of 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 membranes, the size of the nanozymes increased significantly: N-PB nanozyme was 156.5 nm, M-PB nanozyme was 154.1 nm, and NM-PB nanozyme was 159.0 nm.

[0065] Zeta potential measurements showed that compared to unmodified PB (-17.1 mV), the NM-PB nanozymes (-19.0 mV), N-PB nanozymes (-28.4 mV), and M-PB nanozymes (-23.7 mV) exhibited significantly higher negative charges. This phenomenon was attributed to the presence of a strong negative zeta potential on the cell membrane surface, indicating that the PB nanozymes had been successfully modified with the cell membrane, namely encapsulated by the 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 viability assessment

[0069] Fetal human colon (FHC) cells and RAW264.7 cells were seeded in 96-well plates at a density of 7 × 10 cells per well. After culturing for 24 hours in Dulbecco's modified Eagle's medium (DMEM, Gibco, 11965) supplemented with 10% fetal bovine serum (FBS, Procell, 164210), the cells were treated with various concentrations of NM-PB nanozymes for 24 hours. Subsequently, cell viability was assessed using a 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. Referenced]. 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 using CLSM.

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

[0073] Under irradiation with ultraviolet light at a wavelength of 340 nm, titanium dioxide (TiO2) produces hydroxyl radicals (·OH). NM-PB nanozyme (PB concentration of 200 μg / mL) was 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 a BMPO / ·OOH adduct. NM-PB nanozyme (PB concentration of 200 μg / mL) was added to the xanthine / xanthine oxidase system and detected using an ESR spectrometer. In addition, light (500 W bulb) can catalyze the production 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 ESR spectrometer.

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

[0075] FHC cells were cultured in confocal microscopy 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. Intracellular ROS levels were then assessed using DCFH-DA (Biyuntian, S0033), a fluorescent dye commonly used for ROS detection. 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 Taq™ 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 Biotechnology Co., Ltd., China). Protein concentration was determined using the BCA protein assay (P0009, Beyotime Biotechnology Co., Ltd., China). Equal amounts of protein samples (30 μg) were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck KGaA; Darmstadt, Germany). The PVDF membrane was blocked with 5% skim milk at 37°C for 2 hours and then incubated with the primary antibody at 4°C overnight. The PVDF membrane was then incubated with the corresponding secondary antibody for 2 hours at room temperature. Finally, 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, samples were blocked with bovine serum albumin (BSA) for 2 hours and then incubated with primary antibodies (1:200 dilution) overnight at 4°C. The following day, samples were incubated with fluorescent secondary antibodies (1:500 dilution) for 1 hour at room temperature. Samples were then 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 the corresponding doses. During this period, the body weight, stool consistency, and blood in the stool were assessed and recorded daily. Mice were euthanized on day 8, and colon tissues were immediately collected for histological analysis.

[0086] In vivo targeting ability of 1.9NM-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). UC model mice were set as the DSS group, and the mean fluorescence intensity of the colon was calculated. In addition, Dil was used to replace Dir fluorescent dye to label these nanozymes so that their distribution in UC lesions could be observed under a fluorescence microscope.

[0088] 1. In vivo therapeutic effect of 10NM-PB nanozymes

[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). 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 content 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 biochemistry 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 Jiubang Biotechnology Co., Ltd., Quanzhou, Fujian, China).

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

[0093] 1.13 RNA-seq analysis

[0094] C57BL / 6 mice were euthanized, and colon cryosections were obtained for transcriptome analysis. Transcriptome sequencing involves multiple experimental steps, including RNA extraction, sample quality control, library preparation, and sequencing on a sequencing platform. For bioinformatics analysis, the expression level of each transcript was quantified using fragments per kilobase per million mapped reads (FPKM). Data analysis was performed using R software, and 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. Furthermore, identified DEGs were subjected to gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis using Cluster Profiler. Furthermore, expression changes of key genes in these pathways were validated by protein and RNA analysis in colonic tissue.

[0095] 1.14 Statistical Analysis

[0096] Statistical analysis was performed using GraphPad Prism software (version 7.03, GraphPad Software). Data are presented as mean ± standard deviation. 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 < 0.05 was considered statistically significant, and p < 0.01 was considered statistically significant).

[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). Figure 1S In AB, among them, Figure 1SA) shows the survival rate of FHC cells after incubation with NM-PB nanozymes, and B) shows 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 then 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 by the colitis cell model ( Figure 1 F). Apparently, nanozymes modified with two cell membranes possess dual targeting capabilities, thereby enhancing their accumulation 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 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 clear 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 demonstrate that PB nanozymes, which possess antioxidant enzyme activity, can effectively scavenge ROS and protect cells from oxidative stress. Importantly, the antioxidant effect was particularly pronounced in the NM-PB group, suggesting that the dual-targeting capability of the neutrophil-macrophage hybrid membrane enhances the cellular uptake of the PB nanozymes, thereby improving their antioxidant efficacy.

[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 pro-inflammatory phenotype called M1 macrophages, which secrete various pro-inflammatory 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 cytokines associated with inflammation by 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. Notably, NM-PB nanozymes showed a particularly significant anti-inflammatory effect ( 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). These results collectively indicate that NM-PB nanozymes have potent anti-inflammatory capabilities. 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 LPS group. In contrast, the expression of anti-apoptotic protein Bcl2 was upregulated ( Figure 2 J). Notably, among these groups, the NM-PB group exhibited a stronger anti-apoptotic ability. Therefore, it can be inferred that the NM-PB nanozyme 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 cell model.

[0108] WB analysis results showed that the expression of E-cadherin and Occludin was most significantly upregulated in the NM-PB group compared with the PB, N-PB, and M-PB groups ( 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 large amounts 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 to an M2 phenotype, we performed cell immunofluorescence analysis. Our results showed that compared with the LPS group, PB, N-PB, M-PB, and NM-PB nanozymes significantly attenuated LPS-induced M1 macrophage activation and downregulated iNOS expression. 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] These experimental results demonstrate that the NM-PB nanozyme can disrupt the harmful feedback loop established by ROS and excessive immune response in a colitis cell model, thereby alleviating intestinal epithelial cell apoptosis and restoring intestinal mucosal barrier function. Furthermore, it promotes the transformation of M1 macrophages to an M2 phenotype. This effect may be attributed to the dual-targeting ability of the neutrophil-macrophage hybrid membrane, which enhances the intracellular accumulation of the PB nanozyme and further improves its 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 a 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, cited in reference] 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 the inflammatory damage in mice.

[0114] Subsequently, we investigated the effects 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 significantly decreased compared with those in group G2 ( Figure 3 FH). Similarly, 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 can promote apoptosis of intestinal epithelial cells. After treatment with PB nanozymes, we observed a significant downregulation of Bax and an 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] These experimental results demonstrate that PB nanozymes have anti-inflammatory and anti-apoptotic effects and can restore intestinal epithelial barrier function. Given the comparable efficacy observed in the G4 and G5 groups, we selected 20 mg / kg 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 indicators and histopathological examination of important organs. Figure 3S As shown in Figures AF, administration of NM-PB nanozymes did not result in any significant abnormalities in liver function, renal 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 treatment with NM-PB nanozymes ( Figure 3S These findings confirm the complete absorption and metabolism of the NM-PB nanozyme after intravenous injection, demonstrating its favorable biosafety profile. This supports its potential for in vivo applications and provides a viable pathway for therapeutic translation from animal studies to clinical applications.

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

[0120] Ensuring that nanozymes are accurately delivered to the site of 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 focuses on evaluating their localization in UC lesions in mouse models. 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 had significant targeting ability. In addition, we also evaluated the biodistribution of the injected nanozyme 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. 24 hours after intravenous injection, frozen sections of the lesioned intestinal tissue were examined by CLSM. 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 increase in the signal was observed in the NM-PB group ( Figure 4 D), which is consistent with the results of ex vivo fluorescence imaging.

[0122] We also further quantified the enrichment of nanozymes in intestinal lesions by analyzing iron content using inductively coupled plasma optical emission spectroscopy (ICP-OES). Compared with the PB, N-PB, and M-PB groups, the NM-PB group had significantly higher iron concentrations in the lesioned intestinal tissue ( 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 effects were evaluated by assessing the weight changes, disease activity index (DAI), colon length, pro-inflammatory cytokine expression levels, and colon section histological analysis of each group of mice. 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 G6 group was the most obvious. Colonic tissue morphological analysis showed that the colonic crypts in G2 group were severely destroyed, immune cells were widely infiltrated, and colonic epithelial cells were significantly damaged. In contrast, 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 in 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 the cell apoptosis in colon tissue. It is worth noting that the red fluorescence intensity of TUNEL staining in G6 group was significantly reduced compared with 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 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 exploits the inflammatory chemotactic properties of neutrophil and macrophage membranes to promote the accumulation of PB nanozymes in UC lesions. This targeted delivery approach effectively exploits the multiple functions of PB nanozymes, including anti-inflammatory and anti-apoptotic activities, restoration of the intestinal mucosal barrier, and promotion of polarization of M1 macrophages to an M2 phenotype.

[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 these 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 compared with the PBS group, the DSS group had 7008 DEGs, of which 4096 genes were upregulated and 2912 genes were downregulated. After PB nanozyme treatment, a total of 6016 DEGs (2627 upregulated genes and 3389 downregulated genes) were identified. It is worth noting that 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 DEGs to elucidate the therapeutic mechanism of NM-PB nanozymes. Based on the gene ontology (GO) database, 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 the log2FC expression level of DEGs as the evaluation criteria to study the related projects. 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 related to the cytokine-cytokine receptor interaction pathway (Cxcl3, Il19, Il20ra) 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 alleviates the symptoms of the mouse UC model mainly 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 constructed by the present invention, which is wrapped by a neutrophil-macrophage hybrid membrane, aims to achieve targeted treatment of UC and elucidate its underlying molecular mechanism. The hybrid membrane composed of neutrophils and macrophages gives the PB nanozyme dual targeting ability, significantly increasing its accumulation in the UC lesion site. Compared with PB nanozymes wrapped only by neutrophil or macrophage membranes, this design has obvious advantages.

[0133] NM-PB nanozymes demonstrated potent ROS scavenging capabilities in both cellular colitis models and mouse ulcerative colitis models. They effectively inhibited inflammatory responses, reduced apoptosis of intestinal epithelial cells, restored the integrity of the intestinal mucosal barrier, and promoted macrophage polarization from M1 to M2. Furthermore, we found that NM-PB nanozymes could effectively slow the progression of UC in mouse models by inhibiting the cytokine-cytokine receptor interaction signaling pathway. Importantly, NM-PB nanozymes exhibited excellent biocompatibility both in vivo and in vitro. Therefore, NM-PB nanozymes have great potential for 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: The invention relates to 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 according to claim 1, wherein the protein mass ratio of the neutrophil membrane to the 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 biomimetic hybrid membrane-encapsulated Prussian blue nanozyme according to any one of claims 1 to 3, comprising the following steps: 1) Neutrophils and macrophages were collected and membrane protein extraction kits were used 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 an NM hybrid membrane; 3) The NM hybrid membrane and the PB nanozyme were 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 is performed 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 according to any one of claims 1 to 3 in the manufacture of a drug for treating ulcerative colitis.

Images