Metal polyphenol nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to a metal polyphenol nano-enzyme, the metal polyphenol nano-enzyme comprises a core and a shell, the core comprises cerium, tannic acid and resveratrol, the shell comprises a hybrid cell membrane, and the shell wraps the surface of the core. The preparation method of the metal polyphenol nano enzyme comprises the following steps: preparing metal polyphenol nano particles; and extraction of a hybrid cell membrane and integration of a core and a shell. The metal polyphenol nano-enzyme provided by the invention has the advantages that the catalytic activity and selectivity are remarkably improved, the targeting capability and focus enrichment are improved, and controllable release and safety are realized.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a metal polyphenol nanozyme, its preparation method, and its application. Background Technology

[0002] The pathological features of inflammatory bowel disease (IBD) are characterized by a vicious cycle of persistent excessive production of reactive oxygen species (ROS) and inflammatory cascades. This process drives the destruction of the intestinal epithelial barrier, immune cell infiltration, and cytokine storms, ultimately leading to mucosal ulceration and fibrosis. Currently, first-line clinical treatment mainly involves systemic administration of immunosuppressants such as glucocorticoids and 5-aminosalicylic acid. However, long-term, high-dose use is often accompanied by dose-limiting toxic side effects such as systemic immunosuppression, opportunistic infections, osteoporosis, and metabolic disorders, significantly reducing patient adherence and quality of life.

[0003] In recent years, the use of nanozymes to scavenge ROS and restore redox homeostasis in the lesion microenvironment has become a cutting-edge strategy to break through the bottleneck of traditional IBD treatment. Among them, metal-phenolic networks (MPNs) can self-assemble into size-controllable and surface-chemically tunable nanostructures under physiological conditions due to the reversible coordination between polyphenol ligands and metal ions. This type of network has the following advantages: (1) high ROS scavenging activity, which can continuously exert dual functions of catalase-like (CAT) and superoxide dismutase-like (SOD) in complex biological media; (2) good biocompatibility, with the polyphenol skeleton reducing cytotoxicity through weak interactions such as hydrogen bonding and π-π stacking; (3) pH / ROS dual-response degradation characteristics, which ensures on-demand dissociation under the acidic inflammatory microenvironment and high ROS stimulation, avoiding off-target effects in normal tissues.

[0004] Among numerous MPN systems, Ce-tannic acid (Ce-TA) nanozymes stand out. The unique Ce-tannic acid content of cerium ions... 3+ / Ce 4+ Reversible valence cycle endows it with efficient electron transfer capability, enabling continuous catalysis of O2· -→H2O2→H2O cascade reaction; Tannic acid (TA) is rich in pyrogallol structure, which not only acts as a polydentate ligand to stabilize cerium ions, but also further enhances the overall antioxidant efficacy through its own free radical scavenging ability. In addition, the TA-Ce coordination bond strength and pKa value are highly tunable, which can precisely control the redox potential of Ce and achieve a synergistically amplified ROS scavenging effect. However, naked Ce-TA nanoparticles are easily recognized and cleared by the mononuclear-macrophage system after intravenous or oral administration, and have a short circulating half-life; at the same time, they lack the ability to actively target IBD lesions (especially deep ulcers and crypt regions), making it difficult for effective drug concentrations to accumulate at the site of inflammation, thus limiting its in vivo therapeutic index.

[0005] Resveratrol (RSV) is a natural polyphenol with potent ROS scavenging, NF-κB inhibition, and mitochondrial protection effects. However, its extremely low water solubility and first-pass metabolism result in low oral bioavailability, with clinical doses as high as 5g / day. -1 It remains difficult to achieve an effective intestinal concentration. Co-loading Ce-TA nanozymes with RSV can achieve synergistic cascade antioxidant and anti-inflammatory effects, but the challenges of targeted delivery and controlled release still need to be addressed.

[0006] Cell membrane biomimetic technology offers nanomedicines the dual advantages of "self" immune camouflage and active chemotaxis. Macrophage membranes are rich in CCR2 and can infiltrate into the inflammatory base in response to the CCL2 gradient; neutrophil membranes contain CXCR2 and CD11b, which can recognize CXCL1 / 2 and cross the mucus layer and epithelial tight junctions. However, current research is mostly limited to single cell membrane coverage, making it difficult to simultaneously meet the dual targeting requirements across the mucus layer and across the epithelium.

[0007] The existing technologies currently have the following main problems: (1) Targeting limitations: Macrophages themselves have a certain chemotaxis to inflammatory or tumor tissues, but their chemotaxis to IBD lesions (especially the crypt region of ulcerative colitis) is insufficient, making it difficult to overcome the intestinal mucus layer and tight junction barrier, resulting in low delivery efficiency; (2) Insufficient immune escape: Simple macrophage membrane coating cannot effectively escape early immune clearance mediated by neutrophils, and is easily phagocytosed or adhered to by a large number of recruited neutrophils, shortening the circulating half-life; (3) Single catalytic activity: The existing system only uses the metal-polyphenol network as a drug adsorption and photothermal trigger carrier, without utilizing its enzyme-like activity for in-situ ROS clearance; at the same time, divalent metal ions (Cu 2+ Mn 2+(4) Free small molecule RSV has poor water solubility, fast metabolism, and obvious off-target toxicity. (5) Poor release controllability: photothermal triggering requires external laser, which is inaccessible to deep intestinal lesions; and the extracellular adsorption method causes the drug to fall off prematurely under the action of cyclic shear force, resulting in off-target toxicity. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a metal polyphenol nanozyme comprising a core and a shell. The core comprises cerium (Ce), tannic acid (TA), and resveratrol (RSV), and the shell comprises a hybrid cell membrane that wraps around the surface of the core.

[0009] In one embodiment, the hybrid cell membrane includes a macrophage membrane (RM) and a neutrophil membrane (Neu). The raw materials for preparing the metal polyphenol nanozyme include cerium salt, tannic acid, resveratrol, macrophage membrane and neutrophil membrane; the cerium salt includes cerium nitrate (Ce(NO3)3).

[0010] A second aspect of the present invention also provides a method for preparing the above-mentioned metal polyphenol nanozyme, comprising the following steps: Preparation of metal polyphenol nanoparticles: Cerium salt was dissolved in water to obtain a cerium salt solution, tannic acid was dissolved in a solvent to obtain a tannic acid solution, and resveratrol was dissolved in water to obtain a resveratrol solution. The tannic acid solution and resveratrol solution were added dropwise to the cerium salt solution, the pH was adjusted, the mixture was stirred at room temperature in the dark, centrifuged to obtain a precipitate, washed, and freeze-dried to obtain metal polyphenol nanoparticles (Ce-TA-RSV). Extraction of hybrid cell membranes: Macrophages and neutrophils were lysed separately, and the cell membrane precipitates were collected by differential centrifugation. The precipitates were resuspended to obtain macrophage membrane suspensions and neutrophil membrane suspensions. The mixtures were then mixed to obtain a cell membrane mixture. A membrane fusion promoter was added, and the mixture was incubated with shaking. The mixtures were then extruded to obtain hybrid cell membrane vesicles. Core-shell integration: Metal polyphenol nanoparticles and hybrid cell membrane vesicles were mixed and extruded to obtain metal polyphenol nanozymes (Ce-TA-RSV@RN) with a core-shell integrated structure.

[0011] In one embodiment, the preparation of the metal polyphenol nanoparticles includes the following step: sequentially adding tannic acid solution and resveratrol solution to a cerium salt solution.

[0012] In one embodiment, in the preparation of the metal polyphenol nanoparticles, the pH adjustment is performed by adjusting the pH to 6.5-7.5 with NaOH.

[0013] In one embodiment, the extraction of the hybrid cell membrane includes a lysis buffer comprising tris(hydroxymethyl)aminomethane hydrochloric acid solution (Tris-HCl), ethylenediaminetetraacetic acid (EDTA), and a protease inhibitor.

[0014] In one embodiment, during the extraction of the hybrid cell membrane, the pH of the lysing buffer is 7-7.5.

[0015] In one embodiment, the lysis of the hybrid cell membrane is performed in an ice bath for 20-40 minutes.

[0016] In one embodiment, during the extraction of the hybrid cell membrane, the resuspension solution is phosphate-buffered saline (PBS) with a pH of 7-7.5.

[0017] In one embodiment, in the preparation of the metal polyphenol nanoparticles, the solvent includes ethanol and water, with a volume ratio of ethanol:water of (1~2):(1~2).

[0018] In one embodiment, in the preparation of the metal polyphenol nanoparticles, the dropping time is 20-40 min; the room temperature stirring time is 20-40 min; the centrifugation conditions are 10000g-15000g, 8 min-12 min; and the freeze-drying time is 18-30 h.

[0019] In one embodiment, the concentration of the dissolved cerium salt in the preparation of the metal polyphenol nanoparticles is 10-30 mM.

[0020] In one embodiment, the concentration of the dissolved cerium salt is 20 mM during the preparation of the metal polyphenol nanoparticles.

[0021] In one embodiment, the concentration of the dissolved tannic acid in the preparation of the metal polyphenol nanoparticles is 5~20 mg / mL.

[0022] In one embodiment, the concentration of the dissolved tannic acid in the preparation of the metal polyphenol nanoparticles is 10 mg / mL.

[0023] In one embodiment, the concentration of resveratrol after dissolution in the preparation of the metal polyphenol nanoparticles is 5~20 mg / mL.

[0024] In one embodiment, the concentration of resveratrol after dissolution is 10 mg / mL during the preparation of the metal polyphenol nanoparticles.

[0025] In one embodiment, the volume of water used to dissolve the resveratrol in the metal polyphenol nanoparticles is 40-60 mL.

[0026] In one embodiment, the volume of water used to dissolve the resveratrol in the metal polyphenol nanoparticles is 50 mL.

[0027] In one embodiment, the dropwise addition time for the metal polyphenol nanoparticles is 30 min.

[0028] In one embodiment, the room temperature stirring time is 30 min during the preparation of the metal polyphenol nanoparticles.

[0029] In one embodiment, the centrifugation conditions for preparing the metal polyphenol nanoparticles are 12000g for 10min.

[0030] In one embodiment, the centrifugation temperature during the preparation of the metal polyphenol nanoparticles is 23~28°C.

[0031] In one embodiment, the washing solution used in the preparation of the metal polyphenol nanoparticles is phosphate buffered saline (PBS).

[0032] In one embodiment, the freeze-drying time for the preparation of the metal polyphenol nanoparticles is 24 hours.

[0033] In one embodiment, the differential centrifugation in the extraction of the hybrid cell membrane includes: first centrifuging to remove cell debris, and then centrifuging to obtain a membrane precipitate; the membrane fusion promoter includes calcium chloride at a concentration of 0.5~2mM.

[0034] In one embodiment, during the extraction of the hybrid cell membrane, the incubation time is 20-40 min; the hybrid cell membrane vesicle diameter is 90-110 nm.

[0035] In one embodiment, the hybrid cell membrane vesicles have a diameter of 100 nm during extraction.

[0036] In one embodiment, during the extraction of the hybrid cell membrane, the centrifugation conditions for removing cell debris are 600-800g for 8-15min.

[0037] In one embodiment, the centrifugation conditions for obtaining the membrane precipitate during the extraction of the hybrid cell membrane are 10,000-25,000 g for 20-40 min.

[0038] In one embodiment, the centrifugation conditions for obtaining the membrane precipitate during the extraction of the hybrid cell membrane are 15000g for 30min.

[0039] In one embodiment, the concentration of the membrane fusion promoter is 1 mM during the extraction of the hybrid cell membrane.

[0040] In one embodiment, the incubation time for the extraction of the hybrid cell membrane is 30 min.

[0041] In one embodiment, the extraction of the hybrid cell membrane involves continuous extrusion via a polycarbonate membrane extruder.

[0042] In one embodiment, during the extraction of the hybrid cell membrane, the polycarbonate membrane has a pore size of 200-400 nm.

[0043] In one embodiment, the polycarbonate membrane has a pore size of 400 nm during the extraction of the hybrid cell membrane.

[0044] In one embodiment, the polycarbonate membrane has a pore size of 200 nm during the extraction of the hybrid cell membrane.

[0045] In one embodiment, the extraction of the hybrid cell membrane involves 5 to 15 extrusions.

[0046] In one embodiment, the extraction of the hybrid cell membrane involves 11 extrusions.

[0047] In one embodiment, in the core-shell integration, the extrusion is a continuous extrusion via a polycarbonate film extruder.

[0048] In one embodiment, in the core-shell integration, the polycarbonate membrane has a pore size of 100~300nm.

[0049] In one embodiment, in the core-shell integration, the polycarbonate membrane has a pore size of 200 nm.

[0050] In one embodiment, the core-shell integration involves 2 to 5 extrusion cycles.

[0051] In one embodiment, the core-shell integration involves three extrusion cycles.

[0052] In one embodiment, in the preparation of metal polyphenol nanoparticles, the mass ratio of the cerium salt: the tannic acid: the resveratrol is 1:(1~3):(0.1~0.5). In the extraction of hybrid cell membranes, the ratio of macrophage membrane to neutrophil membrane is (1~2):(1~2) according to the protein content ratio. In the core-shell integration, the mass ratio of the metal polyphenol nanoparticles to the hybrid cell membrane vesicles is (1~2):(1~2).

[0053] A third aspect of the present invention also provides the application of the above-described metal polyphenol nanozymes or the metal polyphenol nanozymes obtained by the above preparation method in the preparation of products for treating inflammatory bowel disease.

[0054] Compared with the prior art, the present invention has the following beneficial effects: 1. Significant improvement in catalytic activity and selectivity: (1) Dual cascade enzyme activity: Existing Fe 3+ / Cu 2+ - Polyphenol nanozymes exhibit only a single type of peroxidase activity and require exogenous H2O2 to be effective; this invention utilizes Ce... 3+ / Ce 4+ Valence state cycling endows the complex with SOD+CAT cascade functionality, enabling continuous scavenging of O2· without additional substrate. - (1) With H2O2, the total ROS removal efficiency in vitro is improved; (2) Acid tolerance is enhanced: under the acidity of the inflammatory microenvironment, the Ce-TA coordination network remains dense and intact, and metal ions are almost not free; at the same time, resveratrol is fully released, the drug dosage quickly reaches the required level, and the metal toxicity risk of the system is significantly reduced.

[0055] 2. A leapfrog improvement in targeting ability and lesion enrichment: (1) Dual membrane protein recognition: For the first time, macrophage membrane (CCR2) protein recognition is achieved. + ) and neutrophil membrane (CXCR2) + (1) Hybridization with a 1:1 protein mass ratio to achieve active targeting across the mucus layer and across the epithelium via dual pathways; (2) Extended in vivo circulation cycle: Compared with existing bare nanoparticles, this invention significantly weakens plasma protein adsorption and mononuclear-macrophage system recognition through a macrophage-neutrophil hybrid membrane shell, which significantly extends the residence time of nanoparticles in the blood circulation, and the amount of drug accumulation in the liver and spleen is greatly reduced, resulting in a more lasting and uniform distribution in the body.

[0056] 3. Controllable release and safety advantages: (1) Endogenous triggering: Zero-order kinetic release is achieved by utilizing the dual signals of low pH and high ROS in the lesion, without the need for external laser or magnetic field; approximately 75% is released cumulatively in 48 hours, and <8% in the normal intestine, completely avoiding the problem that existing photothermal triggering schemes cannot reach deep lesions; (2) Reduced systemic toxicity: No abnormalities in liver and kidney tissue pathology were observed after long-term administration. Attached Figure Description

[0057] Figure 1The potential changes of Ce-TA NP, Ce-TA-RSV NP, and Ce-TA-RSV@RN are shown in the diagram. Figure 2 TEM images and particle size distribution of Ce-TA-RSV; Figure 3 The particle size variation of Ce-TA-RSV@RN in different media over 48 hours is shown. Figure 4 Gel electrophoresis images of total protein from RN (a mixture of RAW264.7 macrophages and mouse bone marrow neutrophils), Ce-TA-RSV@RN, and Ce-TA-RSV, where I represents RN (a mixture of RAW264.7 macrophages and mouse bone marrow neutrophils), II represents Ce-TA-RSV@RN, and III represents Ce-TA-RSV. Figure 5 The release curve for RSV; Figure 6 Internalization diagram of Raw246.7 cells treated with Ce-TA-RSV@RN; Figure 7 Cytotoxicity diagrams for Ce-TA NP, Ce-TA-RSV NP, and Ce-TA-RSV@RN; Figure 8 Verification of the effect of ternary eutectic on "TA competition exclusion" and "Ce" using the DPPH radical decolorization method 4+ Absorbance histogram (517nm) showing the effect of overcoming the coupling difficulties of "oxidative deactivation"; Figure 9 The effects of serum biochemical indicators related to liver and kidney function are shown; (A) is serum albumin (ALP), (B) is serum alkaline phosphatase (ALB), (C) is creatinine (CREA), and (D) is urea (UREA). Detailed Implementation

[0058] This invention provides a "dual-drug co-delivery, dual-membrane camouflage ROS cascade clearance nanosystem" with an integrated "core-shell" design: the core is a composite nanoparticle co-loaded with metal-polyphenol nanozyme Ce-tannic acid (Ce-TA) and resveratrol (RSV), and the outer shell is a lipid bilayer formed by hybridization of macrophage membrane (RM) and neutrophil membrane (Neu) in a 1:1 protein ratio. This complex exhibits dual pH / ROS response release characteristics, enabling synergistic ROS clearance by Ce-TA's SOD-CAT cascade and the anti-inflammatory effects of RSV within inflammatory bowel disease lesions. Simultaneously, utilizing the synergistic recognition of membrane proteins RM-CCR2 and Neu-CXCR2, it crosses the mucus layer and epithelial barrier, increasing lesion enrichment and prolonging the blood half-life, significantly outperforming existing single metal-polyphenol or single-membrane encapsulation technologies.

[0059] To address the limitations of existing technologies, such as targeting restrictions, insufficient immune escape, limited catalytic activity, and poor release controllability, this invention primarily solves the following problems: (1) Construct a "macrophage-neutrophil" hybrid membrane coating system. Utilize the chemotaxis of macrophages to the inflammatory basal layer and the high recognition and penetration ability of molecules such as CD11b and CXCR2 on the neutrophil membrane to the intestinal ulcer site. This overcomes the limitations of traditional single macrophage membranes, such as insufficient targeting and difficulty in penetrating the intestinal mucus layer and tight junction barrier, to achieve precise and deep delivery to IBD lesions.

[0060] (2) By hybridizing neutrophil membranes and macrophage membranes in a specific ratio, an immune camouflage layer with expression of both "self" and "non-self" membrane proteins is formed, which significantly reduces the risk of rapid clearance by the host immune system and solves the problem of insufficient immune escape due to simple macrophage membrane coating. This prolongs the in vivo circulation time of nanozymes and increases the accumulation of lesions. In addition to targeting, CD47 and CD55 are expressed on the surface of both membranes, which significantly inhibits complement activation.

[0061] (3) Using Ce-tannic acid (Ce-TA) metal-polyphenol network as the core of nanozyme, fully utilizing Ce 3+ / Ce 4+ The reversible valence cycle brings SOD / CAT bifunctional catalytic activity, while utilizing the free radical scavenging and metal ion chelating capabilities of the TA polyphenol skeleton to achieve cascade clearance of ROS within lesions; and by coordinating regulation to inhibit the dissolution of Ce ions in an acidic inflammatory environment, it solves the problem of existing tannic acid high affinity for resveratrol and Ce... 4+ The invention addresses the three coupled challenges of "oxidative RSV deactivation" and "hydrophobic RSV-induced secondary aggregation." Through low-temperature one-step co-coordination and precise control of molar ratio and pH, it achieves for the first time a ternary eutectic of Ce-tannic acid-resveratrol, overcoming the aforementioned mutual repulsion effects and forming uniform particles suitable for subsequent coating. The ternary network reduces the RSV oxidative degradation rate to a negligible level, breaking through the bottleneck of "insufficient polyphenol protection."

[0062] (4) By utilizing the pH / ROS dual-response degradation characteristics of the metal-polyphenol network, the nanozyme can release Ce-TA active components on demand in inflammatory lesions (low pH, high ROS) and remain stable in the normal intestinal environment (neutral pH, low ROS), thus avoiding drug leakage and off-target toxicity during circulation. This overcomes the shortcomings of existing technologies, such as poor release controllability, the need for external laser triggering, and the inaccessibility of deep lesions.

[0063] (5) One-step low-temperature co-coordination + single gradient centrifugation <90min, there is currently no similar combination of process parameters in the existing technology.

[0064] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0066] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0067] Example This invention provides a method for preparing metal polyphenol nanozymes, the specific steps of which are as follows: (1) Preparation of Ce-TA-RSV nanoparticles First, dissolve 1.736 g of Ce(NO3)3·6H2O in 250 mL of deionized water to a concentration of 20 mM. Separately, dissolve 3.0 g of tannic acid in 300 mL of ethanol / water (1:1, v / v) to a concentration of 10 mg / mL. -1 Dissolve 0.5g of resveratrol in 50mL of water to obtain a concentration of 10mg / mL. -1 The 1L glass beaker was then placed on a magnetic stirrer and stirred continuously at 25°C and 800 rpm. Tannic acid and resveratrol were added dropwise to Ce(NO3)3·6H2O. Immediately after the addition was complete, the pH of the system was adjusted to 7.0 with 0.1M NaOH. The mixture was stirred at room temperature for 30 min, then centrifuged at 12000g for 10 min at 25°C. The supernatant was discarded, and the mixture was resuspended in PBS at 25°C and centrifuged twice. The resulting precipitate was freeze-dried under vacuum for 24 h to obtain a loose powder, which was then stored at 4°C in the dark.

[0068] (2) Extraction of hybrid cell membranes First, macrophage membranes and neutrophil membranes were extracted separately: RAW264.7 macrophages and mouse bone marrow neutrophils were lysed in hypotonic buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4, containing protease inhibitors) on ice for 30 min. Cell debris was removed by differential centrifugation (700 g, 10 min), and the supernatant was then ultracentrifuged at 15000 g for 30 min. The milky white membrane precipitate was collected, resuspended in PBS, and quantified with BCA to obtain a protein concentration of 1 mg / mL for both. -1 Macrophage membrane (RM) and neutrophil membrane (Neu) suspensions were prepared. Subsequently, RM and Neu were mixed at a 1:1 ratio of membrane protein, with a total protein content of 100 mg and a total volume of 100 mL. 1 mM CaCl2 was added to the mixture as a membrane fusion promoter, and the mixture was incubated at 37 °C with gentle shaking for 30 min to allow the two membranes to achieve initial fusion through calcium bridges. Next, the incubated mixed membrane was extruded 11 times continuously through a 400 nm polycarbonate membrane extruder to obtain macrophage-neutrophil hybrid membrane vesicles. Finally, the vesicle size and co-expression of marker proteins such as CCR2, CXCR2, F4 / 80, and CD66b were confirmed by dynamic light scattering and Western blot. The vesicles were then stored at 4 °C for a short period or used immediately for subsequent membrane coating steps.

[0069] (3) Core-shell integration One-step method: pre-made Ce-TA / RSV nanoparticles are co-extruded with double membrane vesicles (200nm filter membrane, 35℃, 3 cycles) to instantly form a core-shell structure; the membrane-core interface achieves a stable bond through the dual effects of metal-phenolic hydroxyl coordination and hydrophobic insertion, and is stable in physiological saline.

[0070] To facilitate understanding of the present invention, the present invention will be described in more detail below with reference to specific embodiments.

[0071] Example 1 Preparation of Ce-TA nanozymes Ce(NO3)3·6H2O was dissolved in deionized water to prepare a 20 mM solution; tannic acid (TA) was dissolved in a mixed solvent of equal volume of ethanol and water to prepare a 10 mg / mL solution; the two solutions were mixed in equal volumes at room temperature with magnetic stirring, and a pale yellow colloid immediately appeared; after stirring for 15 min, the mixture was dialyzed with deionized water through a dialysis bag with a molecular weight cutoff of 10 kDa for 4 h to obtain a purified Ce-TA nanozyme suspension, which was stored at 4 °C protected from light for later use.

[0072] Example 2 Obtaining Ce-TA-RSV co-loaded nanoparticles Resveratrol (RSV) was dissolved in deionized water at a concentration of 10 mg / mL and slowly added dropwise to the Ce-TA suspension obtained in Example 1 (final Ce concentration of 0.5 mM) under stirring. The mixture was stirred at room temperature in the dark for 30 min. The precipitate was then collected by centrifugation (12000 g, 10 min, 25 °C), resuspended in deionized water, and washed once to obtain Ce-TA-RSV composite nanoparticles, which were dispersed in PBS (pH=7.4) for later use.

[0073] from Figure 8 It can be seen that the 40mM Ce(NO3)3·6H2O group has the highest absorbance at 517nm, indicating that TA competition reduces the free RSV, and Ce 4+ Oxidation destroys the hydrogen-donating capacity of RSV, leading to the loss of antioxidant activity activation; however, the absorbance of the present invention is significantly reduced, indicating that low temperature inhibits oxidation and eutectic locks RSV, thus maintaining its DPPH free radical scavenging ability; and the Ce-free group has the lowest absorbance, confirming that RSV's antioxidant capacity is intact when there is no oxidation and no competition.

[0074] Example 3 Extraction of macrophage membranes (RM) RAW264.7 cells were cultured to 90% confluence and washed three times with pre-cooled PBS. Hypotonic lysis buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4, containing protease inhibitors) was added and incubated on ice for 30 min, followed by repeated passing through a 25G needle 20 times. The lysis buffer was centrifuged at 700g for 10 min at 4°C to remove cell debris. The supernatant was then centrifuged at 15000g for 30 min at 4°C. The precipitate was the RM vesicles. After resuspending in PBS, the cells were sterilized by passing through a 0.22 μm filter membrane and quantified using the BCA method.

[0075] Example 4 Extraction of neutrophil membrane (Neu) Mouse bone marrow cells were collected and enriched with granulocytes by Histopaque-1077 gradient centrifugation. Neu vesicles were prepared by hypotonic lysis and differential centrifugation as in Example 3, quantified with BCA, and resuspended in PBS.

[0076] Example 5 Preparation of RM / Neu hybrid membrane The RM and Neu obtained in Examples 3 and 4 were mixed at a protein ratio of 1:1, with a total volume of 1 mL. CaCl2 was added to the mixture as a membrane fusion promoter, and the mixture was placed in a small extruder. The mixture was passed through a 400 nm and a 200 nm polycarbonate membrane three times each to obtain RM / Neu hybrid membrane vesicles. The vesicles were stored at 4 °C and used within 4 hours.

[0077] from Figure 4As can be seen, after total protein separation by SDS-PAGE, the Ce-TA-RSV@RN group and the RN (mixture of RAW264.7 macrophage and mouse bone marrow neutrophil cell membranes) group showed highly similar band distributions in the two lanes within the range of 10-180 kDa, indicating that the protein components of macrophage and mouse bone marrow neutrophil cell membranes were completely preserved and transferred to the surface of the nanocarrier, and there were obvious bands in the range of 40-55 kDa. The hybrid membrane group and the RAW264.7+ neutrophil membrane control group showed bands of consistent intensity at ~47 kDa and ~55 kDa, and their molecular weights were consistent with those of CD47 (47 kDa) and CD55 (55 kDa) reported in the literature, suggesting that the two complement regulatory proteins were completely transferred to the surface of the hybrid membrane.

[0078] Example 6 Construction of Core-Shell Integrated Nanosystems The Ce-TA-RSV particles obtained in Example 2 and the RM / Neu hybrid membrane obtained in Example 5 were mixed at a mass ratio of 1:1, with a total volume of 1 mL. The mixture was then extruded three times through a 200 nm polycarbonate membrane in a water bath at 35 °C to obtain a core-shell nanosystem with "dual drug co-delivery and dual membrane camouflage", denoted as Ce-TA-RSV@RN. The product was stored at 4 °C in the dark for later use.

[0079] Example 7 The performance of the nanoparticles prepared above was verified.

[0080] 1. Potential testing Test method: Dynamic light scattering was used for determination. The specific steps are as follows: Take an appropriate amount of the prepared nanoparticle dispersion, dilute it with ultrapure water to a suitable concentration (0.1 mg / mL), and place it in the sample cell. Use a Malvern nanoparticle size and Zeta potential analyzer to measure at 25℃. Each sample was measured 3 times and the average value was taken.

[0081] Test results: such as Figure 1 As shown in the figure, the Zeta potential of the prepared nanoparticles is... 28.7 ± 1.3 mV indicates that its surface carries a strong negative charge. After being encapsulated by a cell membrane, the Zeta potential of the nanoparticles changes from ( 28.7±1.3)mV shifted to the right to ( 24.3 ± 1.0) mV. The absolute value decreased by approximately 5 mV, indicating that the surface was partially shielded by the electrically neutral film material, maintaining a high negative charge >|25| mV. The aqueous phase dispersion stability was good, and no obvious aggregation was observed. It is generally believed that when the absolute value of the Zeta potential is greater than 30 mV, the system has good stability; however, in this experiment, the Zeta potential was close to... The value of 30mV indicates that the nanoparticles have good dispersion stability in the aqueous phase and are not prone to aggregation.

[0082] 2. Particle size testing and stability testing in different media Test method: Particle size was determined using dynamic light scattering. The specific steps are as follows: Take an appropriate amount of the prepared nanoparticle dispersion, dilute it with ultrapure water to a suitable concentration (0.1 mg / mL), and place it in the sample cell. Measure the particle size and zeta potential using a Malvern nanoparticle size and zeta potential analyzer at 25°C. Each sample was measured three times, and the average value was taken.

[0083] Test results: such as Figure 2 As shown in the figure, the average hydrated particle size of the nanoparticles is 114.70±3.2nm, exhibiting a narrow single-peak distribution.

[0084] Stability testing in different media: Four media (H2O, DMEM, PBS pH=7.4, PBS pH=6.5) were used. The prepared nanoparticles were added to each of the four media and diluted to a final concentration of 0.1 mg / mL. All samples were placed in a 37°C constant temperature shaker for slow shaking incubation to simulate in vivo temperature and flow conditions.

[0085] Small samples were taken at (0h, 6h, 12h, 24h, 48h, 72h) after incubation. The average hydrated particle size was measured using a Malvern nanoparticle size analyzer.

[0086] Test results: In H2O and PBS (pH=7.4), as Figure 3 As shown, the nanoparticles exhibit excellent colloidal stability within 48 hours. The particle size distribution remains flat, with the average particle size maintained at approximately ~115 nm. In PBS (pH=6.5), the particle size reached about 400 nm, indicating that the nanoparticles disintegrated at the site of inflammation, thus releasing the drug.

[0087] 3. Release Test RSV Release Test Method (1) Preparation of test sample Take 1.0 mg of lyophilized Ce-TA-RSV@RN nanozyme and accurately add it to 0.01 mol / L pH 7.4 PBS. -1 1.0 mL, vortexed for 30 s, yields 1 mg / mL -1 Dispersion.

[0088] (2) Release conditions Immediately transfer the above dispersion into a pretreated dialysis bag (MWCO 3kDa), seal the bag tightly, place it in a 15mL centrifuge tube, add 3mL of PBS (pH=6.5 / 7.4) as the external solution, and place the centrifuge tube in a 37°C, 100rpm constant temperature air bath shaker to avoid light.

[0089] (3) Sampling and fluid replenishment At 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours, 1.0 mL of the external solution was taken out and immediately replenished with 1.0 mL of fresh PBS at the same temperature and pH. The collected samples were filtered through a 0.22 µm needle filter and the RSV concentration was determined by UV spectrophotometer.

[0090] (4) Calculation of results Cumulative release percentage (%) = (Qn×3) / (W×LC×1000)×100 Where: W—sample amount of nanozyme (mg); LC—encapsulation amount of RSV in nanozyme (µg mg) -1 ), obtained from the drug loading determination section; 3—total volume of external liquid (mL).

[0091] Test results: such as Figure 5 As shown in the figure, it can be seen that in a medium with pH=6.5, the cumulative release rate of Ce-TA-RSV particles coated with hybrid membranes reached (78.03±73)% in the control group after 6 hours, while that in the Ce-TA-RSV@RN group with pH=7.4 was only (34.79±2)% during the same period. This confirms that the hybrid cell membrane shell can significantly delay the burst release of RSV and achieve pH-responsive continuous release.

[0092] 4. Raw246.7 endocytosis assay RAW264.7 Cell Endocytosis Ce-TA-RSV@RN Assay Method (1) Cell and material preparation RAW264.7 mouse macrophages Complete culture medium: DMEM high glucose + 10% FBS + 1% penicillin and antibiotic-free (2) Flow cytometry for quantitative fluorescence uptake assay Cells were seeded in 24-well plates and incubated overnight at 37°C with 5% CO2. The plates were then replaced with serum-free DMEM, and Ce-TA-RSV@RN pre-labeled with Cy5 (0.5 mg fluorescent dye / 1 mg nanoparticles, incubated at 37°C in the dark for 1, 2, 4, and 6 h, followed by ultrafiltration to remove free dye). The incubation was terminated with PBS (containing 0.02% EDTA) on ice, and cells were collected by trypsin digestion.

[0093] (3) Wash twice with ice-cold PBS, resuspend in 400µL PBS, and then test.

[0094] Test results: such as Figure 6 As shown, the above-mentioned quantitative fluorescence imaging combined method can objectively reflect the changes in fluorescence intensity enhancement over time in the uptake kinetics of Ce-TA-RSV@RN by RAW264.7.

[0095] 5. Cytotoxicity test Assay Method: The cytotoxicity of nanoparticles was assessed using the CCK-8 assay. Raw264.7 cells were seeded at a density of 1 × 10^4 cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. The old culture medium was discarded, and fresh culture medium containing different concentrations of nanoparticles was added, with five replicates for each concentration. A blank control group without cells was also included. After another 24 hours of culture, 10 μL of CCK-8 solution was added to each well, and incubation continued for 4 hours. Subsequently, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%, where As, Ac, and Ab are the absorbance values ​​of the experimental group, control group (without nanoparticles), and blank group, respectively.

[0096] Test results: such as Figure 7 As shown in the figure, cell viability decreased in a dose-dependent manner with increasing nanoparticle concentration. At low concentrations (e.g., 12.5 μg / mL), cell viability remained above 90%, indicating weak cytotoxicity. However, when the concentration increased to 100 μg / mL, cell viability significantly decreased to approximately 60%, demonstrating significant cytotoxicity (p<0.01). At the highest concentration (e.g., 200 μg / mL), cell viability further decreased to approximately 40%, indicating that the nanoparticles had a strong inhibitory effect on Raw264.7 cells at this concentration.

[0097] 6. Serum biochemical markers related to liver and kidney function tests Test Methods: Evaluation was conducted through in vivo animal experiments. C57BL / 6 mice were randomly divided into four groups: PBS, Ce-TANP, Ce-TA-RSV NP, and Ce-TA-RSV@RN. The corresponding formulation was administered via tail vein injection at a dose of 100 μL, every two days for a total of three administrations. Twenty-four hours after the last administration, blood samples were collected from the mice, and serum was separated by centrifugation at 3000 rpm for 15 minutes. Serum levels reflecting liver function (ALB, ALP) and renal function (UREA, CREA) were measured using an automated biochemical analyzer.

[0098] Test results: such as Figure 9As shown, none of the nanoformulations had a significant effect on liver and kidney function in mice. Compared with the PBS group, there were no statistically significant differences in serum ALB, ALP, UREA, and CREA levels in the Ce-TA NP, Ce-TA-RSV NP, and Ce-TA-RSV@RN groups (P>0.05), and all indicators remained within the normal physiological range, suggesting that the constructed nanoplatform has good in vivo biosafety at the experimental dose.

[0099] In summary, the metal polyphenol nanozymes prepared by this invention differ from existing technologies mainly in the following ways: 1. Ternary co-coordinated nanonuclei: In this invention, cerium salt, tannic acid, and resveratrol are added simultaneously at a fixed molar ratio in one step at 4°C to form a dense network with a particle size much smaller than that of traditional systems. Tannic acid acts as both a ligand and a ROS scavenger, while resveratrol is embedded in a co-crystal form, which significantly improves solubility and maintains valence cycle activity. In contrast, existing technologies mostly use divalent copper or trivalent iron to coordinate with a single polyphenol (gallic acid) at room temperature or under heating conditions, resulting in large particle sizes and only peroxidase-like activity, requiring exogenous H2O2; there is no small molecule drug co-embedding.

[0100] Differences: (1) The valence state cycle of the metal center brings about SOD-CAT cascade, which does not require exogenous substrates; (2) The introduction of resveratrol to form a ternary eutectic has significantly better solubility and synergistic anti-inflammatory effects than simple polyphenol networks.

[0101] 2. Macrophage-neutrophil dual-membrane hybrid shell: This invention physically fuses two cell membranes at a protein ratio of 1:1 and then extrudes them to form a continuous double layer with uniform thickness; the surface simultaneously retains CCR2 and CXCR2, achieving dual chemotaxis across the mucus layer and across the epithelium; while existing technologies only use a single macrophage cell membrane or naked particles, lacking neutrophil membrane proteins, and cannot cross the mucus barrier.

[0102] Differences: (1) Dual receptor synergistic targeting, the lesion enrichment multiple is much higher than that of single membrane; (2) Increased bilayer thickness reduces membrane permeability and reduces non-specific adsorption.

[0103] 3. pH / ROS dual-trigger zero-order release: The network of this invention releases the drug at a zero-order uniform rate under the combined action of inflammatory acidity and high ROS, with almost no drug excretion in the normal intestinal environment; while existing technologies rely on tumor high GSH or exogenous photothermal triggering, the trigger signal does not match the IBD lesion, and is prone to pulsed burst release.

[0104] Differences: (1) The triggering source comes from the lesion environment, requiring no additional equipment; (2) The release kinetics are stable, avoiding burst toxicity.

[0105] 4. The carrier-free co-loading mechanism of RSV in metal-polyphenol networks: This invention utilizes Ce 3+ / Ce 4+RSV is directly encapsulated in the coordination network of polyphenol TA; the encapsulation is accomplished solely through π-π stacking, hydrogen bonding, and metal-ligand interactions, without the need for additional polymer or lipid carriers; high drug loading is achieved while simultaneously providing pH / ROS dual-response release; this carrier-free co-loading mechanism can be applied to a universal platform combining any natural small molecule with any metal-polyphenol.

[0106] 5. One-step preparation process: This invention uses low-temperature co-coordination → Ca 2+ Fusion → Single gradient centrifugation can be completed entirely on a conventional magnetic stirrer and standard centrifuge, significantly reducing the total time and minimizing batch-to-batch variation; while existing technologies require column chromatography or multi-step ultracentrifugation, which are time-consuming and have low yields.

[0107] Differences: (1) Simple process; (2) Low equipment requirements and reduced energy consumption.

[0108] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0109] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A metal polyphenol nanozyme, characterized in that, It comprises a core and a shell, the core comprises cerium, tannic acid and resveratrol, and the shell comprises a hybrid cell membrane, and the shell is wrapped on the surface of the core.

2. The metal polyphenol nanozyme according to claim 1, wherein The hybrid cell membrane comprises a macrophage membrane and a neutrophil membrane. The preparation raw materials of the metal polyphenol nanoscale enzyme comprise cerium salt, tannic acid, resveratrol, macrophage membrane and neutrophil membrane; and the cerium salt comprises cerium nitrate.

3. The method for preparing metal polyphenol nanozymes according to claims 1-2, characterized in that, It comprises the following steps: Preparation of metal polyphenol nanoparticles: cerium salt is dissolved in water to obtain a cerium salt solution, tannic acid is dissolved in a solvent to obtain a tannic acid solution, resveratrol is dissolved in water to obtain a resveratrol solution, the tannic acid solution and the resveratrol solution are added dropwise into the cerium salt solution, the pH is adjusted, stirring is carried out at room temperature in the dark, the precipitate is obtained by centrifugation, washing is carried out, and freeze-drying is carried out to obtain metal polyphenol nanoparticles; Extraction of hybrid cell membrane: macrophages and neutrophils are lysed respectively, the cell membrane precipitate is collected by differential centrifugation, and resuspension is carried out to obtain a macrophage membrane suspension and a neutrophil membrane suspension; the cell membrane suspension is mixed to obtain a cell membrane mixture, a membrane fusion promoter is added, and oscillation incubation is carried out to obtain hybrid cell membrane vesicles; Integration of core-shell: the metal polyphenol nanoparticles and the hybrid cell membrane vesicles are mixed and extruded to obtain metal polyphenol nanoscale enzymes with integrated core-shell structure.

4. The production method according to claim 3, characterized by, In the preparation of the metal polyphenol nanoparticles, the dropwise adding comprises: the tannic acid solution and the resveratrol solution are added dropwise into the cerium salt solution in sequence.

5. The preparation method according to claim 3, characterized in that, In the preparation of the metal polyphenol nanoparticles, the solvent comprises ethanol and water, and the volume ratio of ethanol to water is (1-2):(1-2).

6. The preparation method according to claim 3, characterized in that, In the preparation of the metal polyphenol nanoparticles, the dropwise adding is carried out for 20-40 min; the stirring at room temperature is carried out for 20-40 min; the polyvinylpyrrolidone is added and stirring is continued for 10-20 min; the centrifugation is carried out at 10,000 g-15,000 g for 8-12 min; and the freeze-drying is carried out for 18-30 h.

7. The preparation method according to claim 3, characterized in that, In the extraction of the hybrid cell membrane, the differential centrifugation comprises: first centrifuging to remove cell debris, and then centrifuging to obtain membrane precipitate; and the membrane fusion promoter comprises calcium chloride with a concentration of 0.5-2 mM.

8. The preparation method according to claim 3, characterized in that, In the extraction of the hybrid cell membrane, the incubation is carried out for 20-40 min; and the particle size of the hybrid cell membrane vesicles is 90-110 nm.

9. The method of any one of claims 3-9, wherein, In the preparation of the metal polyphenol nanoparticles, the mass ratio of the cerium salt to the tannic acid to the resveratrol is 1:(1-3):(0.1-0.5); In the extraction of the hybrid cell membrane, the mass ratio of the macrophage membrane to the neutrophil membrane is (1-2):(1-2) according to the protein mass; In the integration of the core-shell, the mass ratio of the metal polyphenol nanoparticles to the hybrid cell membrane vesicles is (1-2):(1-2).

10. Use of the metal polyphenol nanoscale enzyme of claim 1-2 or the metal polyphenol nanoscale enzyme obtained by the preparation method of claim 3-9 in the preparation of a product for treating inflammatory bowel disease.

Images