Multistage metal organic framework bionic nano-enzyme loaded with tripterine as well as preparation method and application thereof

Abstract

The invention discloses a multi-stage metal organic framework bionic nano-enzyme loaded with tripterine as well as a preparation method and application thereof, and belongs to the technical field of nano-drugs, the method comprises the following steps: (1) preparing Prussian blue nano-particles by taking potassium ferricyanide, polyvinylpyrrolidone and hydrochloric acid as raw materials; (2) preparing a methanol solution containing Prussian blue nano-particles and polyvinylpyrrolidone, sequentially adding 2-methylimidazole and a zinc source, and reacting to obtain composite nano-particles taking the Prussian blue nano-particles as an inner core and a ZIF-8 aggregation layer as a shell; and (3) loading the tripterine on the ZIF-8 aggregation layer of the composite nanoparticle to obtain a drug-loaded composite nanoparticle, and coating the drug-loaded composite nanoparticle with a macrophage membrane to obtain the multilevel metal organic framework bionic nano-enzyme loaded with the tripterine. The bionic nano enzyme has a wide application prospect in the aspect of treating severe pancreatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-drugs, and in particular relates to a multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii, and a preparation method and application thereof. Background Art

[0002] Severe acute pancreatitis (SAP) is a fatal systemic inflammatory disease, which is often accompanied by mitochondrial dysfunction caused by excessive production of reactive oxygen species (ROS) and impaired autophagic flux. Current clinical treatment strategies mainly focus on single-target intervention models, but the pathological process of SAP involves multiple intertwined pathological links. Although single-dimensional treatment methods can partially alleviate specific pathological stages, their therapeutic effects need to be further improved. SAP is a serious disease with a poor prognosis and a high mortality rate. The development of new therapeutic drugs and treatment strategies is a key direction that needs to be broken through.

[0003] In recent years, nanomedicine has shown good application prospects in the treatment of pancreatitis, especially in the treatment of SAP. It makes up for the shortcomings of traditional treatments through multi-target regulation, precise delivery and synergistic effects, and provides new ideas for the treatment of pancreatitis, especially in controlling severe symptoms and reducing complications. It has breakthrough potential.

[0004] A Chinese patent document with publication number CN118045197A discloses a nanomedicine for targeted treatment of acute pancreatitis. The method first prepares hollow mesoporous Prussian blue nanoparticles, loads a calcium ion chelator blocker and a trypsin inhibitor onto the hollow mesoporous Prussian blue nanoparticles to obtain a complex, wraps the complex with a neutrophil membrane, and then performs phospholipid derivative modification to obtain a nanomedicine for targeted treatment of acute pancreatitis.

[0005] A Chinese patent document with publication number CN117731791A discloses a novel nanomedicine for treating acute pancreatitis and a preparation method thereof. The novel nanomedicine is a multifunctional three-dimensional snowman-like Janus nanoparticle. 2 C and gold nanoparticles as raw materials for the synthesis of Mo 2 C-Au heterojunction was prepared by further coupling gabexate mesylate GM and phospholipid polyethylene glycol amino DSPE.

[0006] The Chinese patent document with publication number CN116832156A discloses a nano drug for treating acute pancreatitis and a preparation method thereof. The invention first prepares a hollow MoS 2 Nanoparticles, in hollow MoS 2 The surface of nanoparticles is modified with gold nanoparticles to obtain Au-MoS 2 Composite nanomaterials, in Au-MoS 2The surface of the composite nanomaterial is modified with lipoic acid-polyethylene glycol to obtain a nanomedicine for treating acute pancreatitis.

[0007] However, none of the above-mentioned nanomedicines can simultaneously treat severe pancreatitis by clearing damaged mitochondria and excess ROS. Summary of the invention

[0008] The present invention provides a multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii and a preparation method thereof. The bionic nanozyme can target inflamed pancreatic tissue, release tripterygium wilfordii in response to an acidic inflammatory microenvironment to promote mitochondrial autophagy, and expose the Prussian blue core to reduce ROS. The above-mentioned combined therapy can simultaneously clear damaged mitochondria and ROS and reduce the expression of inflammatory cytokines, and has broad application prospects in the treatment of severe pancreatitis.

[0009] The specific technical solutions adopted are as follows: A method for preparing a multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii, comprising the following steps: (1) Prussian blue (PB) nanoparticles were prepared using potassium ferrocyanide, polyvinyl pyrrolidone and hydrochloric acid as raw materials. The particle size of the Prussian blue nanoparticles was 50-300 nm. (2) preparing a methanol solution containing Prussian blue nanoparticles and polyvinyl pyrrolidone, adding 2-methylimidazole and a zinc source in sequence for reaction, centrifuging and washing the obtained reaction solution to obtain composite nanoparticles having a core of Prussian blue nanoparticles and a ZIF-8 aggregation layer as an outer shell, wherein the ZIF-8 aggregation layer is composed of ZIF-8 nanoparticles (having a three-dimensional pore network), and the particle size of the composite nanoparticles is 100-500 nm; (3) Loading tripterygium wilfordii on the ZIF-8 aggregation layer of the composite nanoparticles to obtain drug-loaded composite nanoparticles, and coating the drug-loaded composite nanoparticles with macrophage membranes to obtain the multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii.

[0010] The present invention constructs an acid-responsive multi-level metal organic framework bionic nanozyme by growing an aggregation layer shell composed of ZIF-8 nanoparticles on a Prussian blue core. The ZIF-8 aggregation layer carries tripterygium wilfordii to activate mitochondrial autophagy flow. The drug-loaded composite nanoparticles are enclosed by macrophage membranes and have the property of targeting inflammatory pancreatic tissue. Once targeted to the inflammatory area, the drug-loaded composite nanoparticles will be rapidly taken up by pancreatic acinar cells. In the acidic inflammatory microenvironment, acid-induced ZIF-8 degradation will gradually release the loaded tripterygium wilfordii to promote mitochondrial autophagy, while the exposed Prussian blue core is used to reduce ROS. This combined therapy effectively reduces the expression of inflammatory cytokines and restores mitochondrial homeostasis by simultaneously clearing damaged mitochondria and ROS.

[0011] Preferably, in step (1), potassium ferrocyanide and polyvinyl pyrrolidone are added to a hydrochloric acid solution and stirred for 15-20 minutes, the temperature is raised to 75-85° C. and reacted for 18-24 hours, centrifuged, and washed to obtain Prussian blue nanoparticles.

[0012] Further preferably, the mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 1:8-12, and the molar concentration of the hydrochloric acid solution is 0.05-0.15 mol / L.

[0013] Preferably, in step (2), the components include Prussian blue nanoparticles and polyvinyl pyrrolidone in a methanol solution, the concentration of Prussian blue nanoparticles is 0.95-1.05 mg / mL, and the concentration of polyvinyl pyrrolidone is 7-8 mg / mL; in the process of preparing the composite nanoparticles, 42-44 mmol / L of 2-methylimidazole solution is first added for reaction, and then 42-44 mmol / L of Zn(NO 3 ) 2 Solution reaction.

[0014] Further preferably, the components include Prussian blue nanoparticles and a methanol solution of polyvinyl pyrrolidone, a 2-methylimidazole solution and Zn(NO 3 ) 2 The volume ratio of the solution is 1:1.5-1.75:0.2-0.4.

[0015] Preferably, in step (3), a celastrol solution is added dropwise to a methanol solution of the composite nanoparticles to form an indigo mixture, which is stirred at room temperature for 18-24 h, centrifuged, and washed to obtain drug-loaded composite nanoparticles; the mass ratio of the composite nanoparticles to celastrol is 1:0.1-1, and more preferably 1:0.15-0.25.

[0016] Preferably, in step (3), when the drug-loaded composite nanoparticles are coated with macrophage membranes, the weight ratio of the macrophage membranes to the drug-loaded composite nanoparticles is 1:2-2.5.

[0017] The present invention also provides a multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii obtained by the preparation method of the multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii.

[0018] The multi-level metal organic framework bionic nanozyme structure loaded with tripterygium wilfordii comprises a macrophage membrane and drug-loaded composite nanoparticles coated in the macrophage membrane, the drug-loaded composite nanoparticles comprising a Prussian blue nanoparticle core and a ZIF-8 aggregation layer shell, and the ZIF-8 aggregation layer is loaded with tripterygium wilfordii.

[0019] The present invention also provides a drug for treating severe pancreatitis, comprising the multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) In the multi-level metal-organic framework bionic nanozyme loaded with tripterygium wilfordii, the drug-loaded composite nanoparticles are enclosed by the macrophage membrane, which has the property of targeting inflamed pancreatic tissue, has good anti-inflammatory effect, and has low toxicity to normal tissues.

[0021] (2) The present invention greatly improves the solubility of tripterygium wilfordii in water by loading tripterygium wilfordii on the ZIF-8 aggregation layer, making its in vivo application possible.

[0022] (3) The multi-level metal-organic framework biomimetic nanozyme loaded with tripterygium wilfordii targets the inflammatory area and is rapidly taken up by pancreatic acinar cells after reaching the inflammatory area. In the acidic inflammatory microenvironment, acid-induced degradation of ZIF-8 gradually releases the loaded tripterygium wilfordii to promote mitochondrial autophagy, while the exposed Prussian blue core is used to reduce ROS. This combined therapy can effectively reduce the expression of inflammatory cytokines and restore mitochondrial homeostasis by simultaneously clearing damaged mitochondria and ROS, thereby achieving the effect of treating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM images of PB nanoparticles, PBZ nanoparticles, PBZC nanoparticles and multi-level metal organic framework bionic nanozymes (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1.

[0024] Figure 2 This is a graph showing the particle sizes of PB nanoparticles, PBZ nanoparticles, PBZC nanoparticles, and MPBZC nanoparticles in Example 1.

[0025] Figure 3 : This is the potential diagram of PB nanoparticles, PBZ nanoparticles, PBZC nanoparticles and MPBZC nanoparticles in Example 1.

[0026] Figure 4 This is a particle size diagram of the multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1 under different pH conditions.

[0027] Figure 5 This is a statistical graph of the average fluorescence intensity of cells after administration of the multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1, where *** indicates p <0.001, discrete points represent the data values ​​of parallel experiments.

[0028] Figure 6 This is a confocal imaging fluorescence intensity statistical diagram for evaluating the use of the multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1 for restoring mitochondrial function, where *** indicates p <0.001, discrete points represent the data values ​​of parallel experiments.

[0029] Figure 7 Inflammatory factor level diagram for evaluating the therapeutic effect of multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii on severe pancreatitis in Example 1, where * indicates p <0.05,** indicates p <0.01, *** indicates p <0.001, discrete points represent the data values ​​of parallel experiments. DETAILED DESCRIPTION

[0030] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0031] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field.

[0032] Example 1

[0033] (1) Synthesis of Prussian blue (PB) nanoparticles 1 g potassium ferrocyanide and 10 g PVP-K30 (polyvinyl pyrrolidone-K30) were added to 80 mL 0.1 mol / L hydrochloric acid solution, stirred at room temperature for 15 min, and the resulting clear solution was heated to 80 °C and stirred for 20 h. After the solution was cooled to room temperature, the reaction solution was diluted to a volume greater than 800 mL. After dilution, the reaction solution was centrifuged at 20,000 rpm / min for 15 min, the precipitate was washed with deionized water 3 times and dried overnight to obtain dark blue powdered Prussian blue nanoparticles (denoted as PB, 1.5 g).

[0034] (2) Synthesis of PB-ZIF-8 (PBZ) nanoparticles 10 mg of Prussian blue nanoparticles and 75 mg of PVP-K30 were dissolved in 10 mL of methanol, and the mixture was ultrasonicated in an ice bath (300 W, 2s / 2s) for 30 min. Then, the resulting clear solution was stirred at 300 rpm / min, and 16 mL of 43 mmol / L 2-methylimidazole methanol solution was added. After the reaction solution was stirred at room temperature for 15 min, 4 mL of 43 mmol / L Zn(NO 3 ) 2 6H 2 O aqueous solution, and stirred at room temperature for another 60 min. The resulting solution was centrifuged at 13000 rpm / min for 30 min at room temperature, and the precipitate was washed with methanol three times to obtain PB-ZIF-8 (PBZ) composite nanoparticles. The resulting PBZ nanoparticles were dispersed in methanol and stored at 4°C in the dark.

[0035] (3) Synthesis of PBZ-triptertium wilfordii (PBZC) nanoparticles 5 mL (1 mg / mL) of PBZ nanoparticles were diluted with 10 mL of methanol, and then 0.1 mL (10 mg / mL) of celastrol methanol solution was slowly added dropwise to form an indigo mixture. After vigorous stirring at room temperature for 24 hours, the mixture was centrifuged at 13,000 rpm / min for 30 min at 4 °C, and the precipitate was washed three times with methanol to obtain PBZ-celastrol (PBZC) nanoparticles. The obtained nanoparticles were dispersed in water and stored at 4 °C for subsequent use.

[0036] (4) Synthesis of macrophage membrane-coated PBZC (MPBZC) nanoparticles Macrophage RAW264.7 cells were gently washed three times with pre-cooled PBS and then centrifuged at 3000 rpm / min for 15 min. The collected cells were resuspended with Cell Membrane Protein Extraction Reagent A (P0033, Beyotime Biotech), incubated at 4 °C for 30 min, and then sonicated in an ice bath for 30 min (300 W, 2s / 2s). The mixture was then centrifuged at 3000 g for 5 min at 4 °C, and the supernatant (containing cell membranes) was collected. The supernatant was pushed through a 400 nm polycarbonate membrane for 7 cycles using an Avanti mini extruder. The supernatant was then centrifuged at 150,000 g for 2 h in an Optima XPN-100 ultracentrifuge to obtain cell membranes. The obtained cell membrane concentration was then measured using an enhanced BCA protein assay kit (P0009, Beyotime Biotech). The obtained cell membrane was mixed with PBZC nanoparticles in a weight ratio of 1:2, and then ultrasonicated (30 W, 2s / 2s) for 10 min in an ice bath to promote cell membrane to wrap around the nanoparticles. Finally, the reaction solution was centrifuged at 3000 g for 5 min at 4 °C to remove excess cell membrane fragments, and finally macrophage membrane-coated PBZC nanoparticles were obtained, which were multi-level metal organic framework bionic nanozyme MPBZC loaded with tripterygium wilfordii.

[0037] Example 2

[0038] The only difference between the preparation method of the multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii in this embodiment and that in Example 1 is that the mass ratio of the composite nanoparticles to tripterygium wilfordii is 1:0.1.

[0039] Example 3

[0040] The only difference between the preparation method of the multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii in this embodiment and that in Example 1 is that the mass ratio of the composite nanoparticles to tripterygium wilfordii is 1:0.5.

[0041] Example 4

[0042] The only difference between the preparation method of the multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii in this embodiment and that in Example 1 is that the mass ratio of the composite nanoparticles to tripterygium wilfordii is 1:1.

[0043] Sample analysis Figure 1-Figure 3The transmission electron microscope images, particle size images and potential images of the PB nanoparticles, PBZ nanoparticles, PBZC nanoparticles and multi-level metal organic framework bionic nanozymes (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1 respectively show that the PB nanoparticles were successfully synthesized, the particle size of the PB nanoparticles was 50-300 nm, and the PBZ nanoparticles included a Prussian blue nanoparticle core and a ZIF-8 aggregation layer shell, the ZIF-8 aggregation layer was composed of ZIF-8 nanoparticles, and the particle size of the PBZ nanoparticles was 50-300 nm; after loading with tripterygium wilfordii, the potential of the drug-loaded composite nanoparticles changed, and the MPBZC nanoparticles included macrophage membranes and drug-loaded composite nanoparticles coated in the macrophage membranes.

[0044] Figure 4 The particle size diagram of the multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1 under different pH conditions. The results show that changes in pH values ​​will cause the deconstruction of the nanoparticles, thereby releasing the drug.

[0045] Anti-inflammatory validation of the multi-level metal organic framework bionic nanozyme (MPBZC nanoparticles) loaded with tripterygium wilfordii in Example 1: C57BL / 6 male mice (6-8 weeks old, 20-25 g) and ICR female mice (4-5 weeks old, 14-16 g) were provided by the Animal Experiment Center of the Run Run Shaw Hospital, School of Medicine, Zhejiang University. All animal experiments were performed in accordance with the guidelines of the Animal Welfare and Ethics Committee of Zhejiang University (approval number: SRRSH202402274).

[0046] In the cholecystokinin-induced acute pancreatitis model, mice were treated with intraperitoneal injection of cholecystokinin (50 μg / kg / h) once every hour for seven consecutive injections, and the last injection was co-administered with lipopolysaccharide (LPS; 10 mg / kg; Solebol). Mice were killed 12 hours after the first injection, and blood and tissue samples were collected. The mice were divided into six experimental groups (NC group, Model group, PB group, CEL group, MPBZC group, and PB+CEL group). The NC group was a healthy control, and the other five groups were treated with PBS, PB nanoparticles (2.5 mg / kg), free tripterygium wilfordii CEL (1 mg / kg), MPBZC (equivalent concentration [PB] = 2.5 mg / kg, equivalent concentration [CEL] = 1 mg / kg), and CEL+PB (equivalent concentration [PB] = 2.5 mg / kg, equivalent concentration [CEL] = 1 mg / kg) via tail vein injection.

[0047] To obtain serum, whole blood samples collected from mice were centrifuged at 3000 rpm for 20 min. The enzymatic activities of amylase and lipase and the levels of inflammatory cytokines in serum were quantitatively detected using commercial kits. Figure 5-Figure 7 (GCLM stands for glutamine-cysteine ​​ligase, and GSS stands for glutathione) It can be seen that MPBZC nanoparticles can effectively inhibit the release of inflammatory factors such as TNF-α, IL-6 and IL-1β, and play a role in controlling pancreatitis.

[0048] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a multi-stage metal organic framework bionic nanozyme loaded with tripterygium wilfordii, characterized in that: The following steps are involved: (1) Prussian blue nanoparticles were prepared using potassium ferrocyanide, polyvinyl pyrrolidone and hydrochloric acid as raw materials. The particle size of the Prussian blue nanoparticles was 50-300 nm. (2) preparing a methanol solution containing Prussian blue nanoparticles and polyvinyl pyrrolidone, adding 2-methylimidazole and a zinc source in sequence for reaction, centrifuging and washing the obtained reaction solution to obtain composite nanoparticles having a core of Prussian blue nanoparticles and a ZIF-8 aggregation layer as an outer shell, wherein the ZIF-8 aggregation layer is composed of ZIF-8 nanoparticles, and the particle size of the composite nanoparticles is 100-500 nm; (3) Loading tripterygium wilfordii on the ZIF-8 aggregation layer of the composite nanoparticles to obtain drug-loaded composite nanoparticles, and coating the drug-loaded composite nanoparticles with macrophage membranes to obtain the multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii.

2. The method for preparing a multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 1, characterized in that: In step (1), potassium ferrocyanide and polyvinyl pyrrolidone are added to a hydrochloric acid solution and stirred for 15-20 minutes, the temperature is raised to 75-85° C. and reacted for 18-24 hours, centrifuged, and washed to obtain Prussian blue nanoparticles.

3. The method for preparing the multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 2, characterized in that: The mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 1:8-12, and the molar concentration of the hydrochloric acid solution is 0.05-0.15 mol / L.

4. The method for preparing a multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 1, characterized in that: In step (2), the components include Prussian blue nanoparticles and polyvinyl pyrrolidone in a methanol solution, the concentration of Prussian blue nanoparticles is 0.95-1.05 mg / mL, and the concentration of polyvinyl pyrrolidone is 7-8 mg / mL; in the process of preparing the composite nanoparticles, firstly add 42-44 mmol / L of 2-methylimidazole solution for reaction, and then add 42-44 mmol / L of Zn(NO3)2 solution for reaction.

5. The method for preparing a multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 4, characterized in that: The components include Prussian blue nanoparticles and methanol solution of polyvinyl pyrrolidone, 2-methylimidazole solution and Zn(NO3)2 solution in a volume ratio of 1:1.5-1.75:0.2-0.

4.

6. The method for preparing a multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 1, characterized in that: In step (3), the celastrol solution is added dropwise to the methanol solution of the composite nanoparticles to form an indigo mixture, which is stirred at room temperature for 18-24 h, centrifuged, and washed to obtain drug-loaded composite nanoparticles; the mass ratio of the composite nanoparticles to celastrol is 1:0.1-1.

7. The method for preparing a multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 1, characterized in that: In step (3), when the drug-loaded composite nanoparticles are coated with macrophage membranes, the weight ratio of the macrophage membranes to the drug-loaded composite nanoparticles is 1:2-2.

5.

8. A multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii obtained according to the preparation method of the multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii according to any one of claims 1 to 7.

9. The multi-stage metal organic framework biomimetic nanozyme loaded with tripterygium wilfordii according to claim 8, characterized in that: The structure includes a macrophage membrane and drug-loaded composite nanoparticles coated in the macrophage membrane. The drug-loaded composite nanoparticles include a Prussian blue nanoparticle core and a ZIF-8 aggregation layer shell. The ZIF-8 aggregation layer is loaded with tripterygium wilfordii.

10. A drug for treating severe pancreatitis, characterized in that: It comprises the multi-level metal organic framework bionic nanozyme loaded with tripterygium wilfordii as described in claim 8 or 9.

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