Cascade catalytic reactor based on Pickering emulsion and preparation method and application thereof
By assembling uridase and catalase at the Pickering emulsion interface, combined with DSPE-PEG-Mannose targeting molecules, the prepared cascaded catalytic reactor solves the complexity of enzyme assembly, and achieves efficient removal of MSU crystals and ROS, significantly improving the symptoms of gouty arthritis.
Patent Information
- Application Number
- CN202510580856.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-01
AI Technical Summary
In the prior art, the assembly methods of uridase and catalase are complex, which may lead to the destruction of enzymes. Unreasonable assembly will hinder the accessibility of active sites and the transmission of intermediate products, reduce catalytic efficiency, and at the same time, there is a lack of self-cascaded catalytic reactors for regulating inflammatory reactions.
The active uridase and catalase were assembled at the oil-water interface by using the Pickering emulsion interface, combined with DSPE-PEG-Mannose as targeting molecules, and a cascaded catalytic reactor was prepared by a one-step ultrasonic method, and methotrexate was encapsulated to inhibit ROS and inflammatory cytokines.
The stability and activity of enzymes are maintained, the catalytic efficiency of cascade is improved, the MSU crystals are significantly cleared, the ROS is reduced, the macrophage polarization is promoted, and the symptoms of gouty arthritis is improved, providing a simple and effective treatment platform.
Smart Images
Figure CN120393049A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to a Pickering emulsion-based cascade catalytic reactor, a preparation method thereof, and an application thereof. Background Art
[0002] Gouty arthritis (GA) is one of the most common inflammatory diseases. The clinical features of GA are manifested as redness, swelling, stiffness and even joint deformity of the toe joints, accompanied by severe pain, which severely damages the physical and mental health of patients. Research shows that when the production of uric acid in the body increases and the excretion is insufficient, monosodium urate (MSU) crystals can accumulate around the joints. MSU crystals promote the activation of local immune cells and produce inflammatory factors, resulting in a large number of neutrophils infiltrating into the joint cavity and around the synovium, and releasing excessive hydrogen peroxide (H2O2) and superoxide anion (O 2− ), etc. Reactive oxygen species (ROS), triggering oxidative stress and inflammatory signal cascade amplification effects, resulting in the imbalance of the immune microenvironment in the joint cavity. If not effectively controlled for a long time, it will cause the activation disorder of inflammatory M1 macrophages, exacerbate tissue damage, and even tissue degradation.
[0003] Uricase (Uri) is an enzyme that catalyzes the degradation of uric acid into allantoin, plays an important role in rapidly reducing the uric acid level in the body, and partially degrades monosodium urate (MSU) crystals. However, in this catalytic process, uricase generates H2O2 as a by-product, resulting in chemical toxicity. Therefore, timely removal of H2O2 is crucial in uricase-based therapy. Components (proteases and nanozymes) with catalase (CAT) activity can remove H2O2, reduce cytotoxicity, and promote cascade catalytic reactions. Existing nanoparticles, liposomes, etc. can simultaneously deliver components with uricase and catalase activity. However, the preparation methods of these technologies usually involve complex chemical reactions, which may cause damage to the enzymes, and there are challenges in simultaneously assembling other immune molecules to regulate the inflammatory microenvironment. In addition, the unreasonable assembly of uricase and components with catalase activity may hinder the accessibility of active sites and the transport of intermediate products, ultimately reducing the catalytic efficiency.
[0004] Pickering emulsion interfacial catalysis provides a new perspective for catalytic reactions in modern synthetic chemistry. Its unique interfacial properties play an important role in controlling enzyme specificity, preventing interference between enzyme-catalyzed reaction networks, and promoting the transport of reaction intermediates. Currently, there is no research on preparing a Pickering emulsion-based self-cascade catalytic reactor using uricase and protease or nanozyme with uricase activity as stabilizers. There is also no report on using the oil phase of the self-cascade catalytic reactor to encapsulate methotrexate for regulating inflammatory reactions, nor is there a report on modifying the surface of the self-cascade catalytic reactor with DSPE-PEG-Mannose as a targeting molecule for macrophages. Summary of the Invention
[0005] In view of the existing R & D gaps in the prior art, the present invention provides a cascade catalytic reactor based on Pickering emulsion.
[0006] The present invention also provides a preparation method for the above-mentioned cascade catalytic reactor based on Pickering emulsion.
[0007] Another object of the present invention is to provide the application of the above-mentioned cascade catalytic reactor based on Pickering emulsion.
[0008] The technical solution adopted by the present invention to achieve the above object is as follows: The present invention provides a cascade catalytic reactor based on Pickering emulsion, which is composed of Uri and CAT active ingredients, methotrexate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyvinyl alcohol-mannose (DSPE-PEG-Mannose).
[0009] Preferably, the cascade catalytic reactor is composed of the following raw materials in parts by mass: Catalase (CAT) active ingredient: 500 - 10000 parts; Uricase (Uri): 500 - 10000 parts; Squalene: 20 - 200 parts; Methotrexate: 10 - 50 parts; DSPE-PEG-Mannose: 1 - 10 parts.
[0010] Preferably, the cascade catalytic reactor further includes 200 - 1000 parts of ultrapure water and 0.1 - 1 mL of PBS buffer solution with a concentration of 50 - 160 mM and a pH of 7.4 - 8.0.
[0011] Preferably, the catalase active ingredient is a nanozyme or a protease.
[0012] Preferably, the nanozyme is MnO2 nanozyme, Pt nanozyme, Pd nanozyme or CeO2 nanozyme; the protease is catalase.
[0013] The present invention also provides a preparation method of the above cascade catalytic reactor, comprising the following steps: (1) Disperse the prescription amount of catalase active ingredient in ultrapure water and stir to obtain liquid A; (2) Disperse the prescription amount of uricase in ultrapure water to obtain liquid B; (3) Disperse the prescription amount of DSPE-PEG-Mannose in ultrapure water to obtain liquid C; (4) Disperse the prescription amount of methotrexate in squalene to obtain liquid D; (5) Mix liquid A, liquid B, and liquid C, ultrasonically mix for 5 seconds, then add liquid D and ultrasonically emulsify to obtain a self-cascade catalytic reactor based on Pickering emulsion; or (a) Disperse uricase and catalase active ingredient in a buffer solution to obtain liquid A; (b) Disperse DSPE-PEG-Mannose in ultrapure water to obtain liquid B; (c) Disperse methotrexate in squalene to obtain liquid C; (d) Mix liquid A and liquid B solutions, ultrasonically mix, then add liquid C and continue ultrasonication to obtain a self-cascade catalytic reactor based on Pickering emulsion.
[0014] Preferably, in step (1), the stirring time is 10 - 60 min; in step (5), the ultrasonication time is 1 - 5 min.
[0015] Preferably, in step (d), the ultrasonication mixing time is 5 - 10 s; the continued ultrasonication time is 1 - 5 min.
[0016] Another object of the present invention is to provide the application of the above cascade catalytic reactor in the preparation of drugs for treating gouty arthritis.
[0017] The present invention utilizes the assembly of uricase and components with catalase activity at the oil-water interface, which can enhance the self-cascade catalytic reaction without the need for additional mediators to alleviate inflammatory diseases. Herein, uricase and catalase-like components are arranged at the oil-water interface to prepare Pickering emulsions, which can serve as self-cascade catalytic reactors, exhibiting excellent cascade catalytic ability and H2O2 scavenging potential. The tight adsorption of enzyme components at the interface reduces the interfacial tension and increases the stability of the self-cascade catalytic reactor. In addition, Pickering emulsions have a multi-level structure. By using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyvinyl alcohol (DSPE-PEG), a lipid-polymer conjugate, mannose ligands are introduced onto the surface of the self-cascade catalytic reactor to enhance the affinity and uptake of macrophages. Methotrexate encapsulated in squalene (as the oil phase) inhibits the production of ROS and inflammatory cytokines, thus promoting macrophage polarization. Notably, the self-cascade catalytic reactor can be synthesized by a one-step sonication method, avoiding complex chemical reactions and maintaining the activity of each component. We predict that the self-cascade catalytic reactor can exhibit excellent biocompatibility and safety, effectively remove MSU deposits and reprogram the inflammatory microenvironment, making it an ideal platform for the treatment of GA.
[0018] The beneficial effects of the present invention are as follows: (1) The self-cascade catalytic reactor provided by the present invention is prepared by simultaneously assembling uricase and catalase active components onto the oil-water interface. The two enzymes can be proportioned in different ratios to improve the cascade catalytic efficiency and reduce oxidative stress; (2) Nanoparticles can simultaneously deliver uricase and catalase active components, but it is still a great challenge to load poorly water-soluble methotrexate at a suitable spatial position. The Pickering emulsion provided by the present invention has a multi-level structure (particles, oil-water interface and oil core) that can load multiple components. Methotrexate can be encapsulated inside to achieve the delivery of multiple components and reduce toxicity and side effects. The preparation of the self-cascade catalytic reactor does not require complex chemical reactions and multi-step assembly. Only through a one-step sonication method, targeted molecules, enzyme molecules and immunomodulators can be orderly assembled to achieve the integration of targeted therapy, MSU crystal degradation and immune regulation; (3) The self-cascade catalytic reactor provided by the present invention, under the reasonable assembly of various components, can completely remove MUS crystals in the joint cavity and cells, and significantly reduce ROS, polarize M1 cells into M2 cells, and completely reverse the immune state of the inflammatory microenvironment. The self-cascade catalytic reactor alleviates joint swelling, improves gait limp and promotes tissue repair in vivo, showing the potential to be an effective therapeutic dosage form for the treatment of gouty arthritis, and providing a new strategy for the development of bioreactors with enzyme cascade catalysis and multi-component synergistic effects.
[0019] (4) The preparation method provided by the present invention is simple and highly controllable. The Pickering emulsion interface prepared can increase the transfer efficiency of intermediate products and promote the cascade catalysis effect. At the same time, DSPE-PEG-Mannose significantly enhances the targeting effect of the emulsion, and methotrexate inhibits cell oxidative stress. Each component can synergistically treat gouty arthritis. Meanwhile, this method can avoid complex chemical reactions and maintain the activity of each component. Description of the Drawings
[0020] Figure 1 Characterization of the self-cascade reactor PEMU prepared in Example 1; wherein: A. Optical micrograph of PEMU, B. Particle size distribution of PEMU, C. Zeta potential distribution of PEMU, D. MSU crystal clearance rate, E. O2 generation amount; Figure 2 Characterization of the self-cascade reactor PECU prepared in Example 2; wherein: A. Optical micrograph of PECU, B. Particle size distribution of PECU, C. Zeta potential distribution of PECU, D. MSU crystal clearance rate, E. O2 generation amount; Figure 3 Effect of the self-cascade catalytic reactor prepared in Example 2 on scavenging ROS; Figure 4 Evaluation of the therapeutic effect of GA; wherein, A. Schematic diagram of GA modeling and treatment protocol, B. Histological evaluation of synovial pathology in GA mice treated with PECU. Detailed Embodiments
[0021] The technical solutions of the present invention will be further explained and illustrated below through specific examples. It should be understood that these examples are only for specific illustration and not as a limitation to the scope of the present invention.
[0022] DSPE-PEG-mannose (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyvinyl alcohol-mannose) used in the present invention is commercially available and purchased from Weihua Biotech.
[0023] Example 1 Reactor with self-cascade catalysis: 3000 parts of ultrapure water 1, 7000 parts of potassium permanganate, 12000 parts of absolute ethanol; 800 parts of MnO2 nanozyme, 800 parts of Uri, 30 parts of squalene, 2.5 parts of DSPE-PEG-Mannose, 200 parts of ultrapure water 2, 50 parts of ultrapure water 3, and the buffer solution is phosphate buffer solution, 100 mM, pH 8.0.
[0024] The preparation method is as follows: (1)Preparation method of nanozyme: 100,000 parts of bovine serum albumin 1 are dispersed in 3,000 parts of ultrapure water 1. At room temperature, 12,000 parts of absolute ethanol and 2 parts of glutaraldehyde are slowly added and stirred for crosslinking for 1 hour to obtain liquid A; 7,000 parts of potassium permanganate are dissolved in 3,000 parts of ultrapure water 1 to obtain liquid B. When liquid A turns slightly milky white, liquid B is immediately added to liquid A, and the mixture is stirred at room temperature for 1.5 hours to obtain a brown solution. Then, vacuum rotary distillation is carried out at 40 °C, and 1,000 parts of ultrapure water 2 are added for dispersion every 1 minute. When no liquid is distilled out, the mixed solution is taken out, centrifuged at 15,000 rpm for 10 minutes to obtain a brown precipitate. After washing twice with 3,000 parts of ultrapure water 1, it is centrifuged at 10,000 rpm for 10 minutes to take the supernatant, and freeze-dried to obtain MnO2 nanozyme.
[0025] (2)Preparation of self-cascade catalytic reactor (PEMU) based on Pickering emulsion: Weigh the prepared MnO2 nanozyme and disperse it in 200 parts of buffer solution to obtain liquid A; disperse 800 parts of Uri in 200 parts of ultrapure water 2 to obtain liquid B; disperse 2.5 parts of DSPE-PEG-Mannose in 50 parts of ultrapure water 3 to obtain liquid C. Mix 2,000 parts of solutions A, B, and C, and mix them evenly by ultrasonic treatment for 5 seconds. Then add 30 parts of squalene, and after mixing, perform ultrasonic treatment for 3 minutes to obtain a self-cascade catalytic reactor.
[0026] A mixed system (PEU + PEM) of Uri-stabilized Pickering emulsion (PEU) and MnO2-stabilized emulsion (PEM) is prepared in a similar way as above.
[0027] Figure 1 Characterization of the self-cascade reactor PEMU prepared in Example 1; among them: A. Light microscopy image, B. Particle size, and C. Zeta potential distribution show that PEMU can be successfully prepared by ultrasonic emulsification method, with good dispersibility and uniform droplet size; D. Catalytic reaction shows that compared with the free enzymes (Uri + MnO2 nanozyme), the mixed system (PEU + PEM) of Uri-stabilized Pickering emulsion (PEU) and MnO2-stabilized emulsion (PEM), the degradation efficiency of PEMU for MSU crystals is significantly improved; in addition, E. O2 generation amount shows that the amount and rate of O2 generated by PEMU degrading H2O2 are significantly increased, indicating that the self-cascade reactor prepared by using MnO2 nanozyme and Uri significantly improves the cascade catalytic performance, providing the possibility for the complete removal of MSU crystals in the joint cavity.
[0028] Example 2 A liquid with a self-cascading catalytic reactor contains 800 parts of catalase, 800 parts of uricase, 30 parts of squalene, 15 parts of methotrexate, 2.5 parts of DSPE-PEG-Mannose, 400 parts of ultrapure water 1, 50 parts of ultrapure water 2, and the buffer solution is phosphate buffer, with a concentration of 100 mM and a pH of 8.0.
[0029] The specific preparation method is as follows: Preparation of Uri and CAT-stabilized self-cascading catalytic reactor (PECU): Weigh 800 parts of Uri and 800 parts of CAT and disperse them in 400 parts of buffer solution to obtain liquid A; disperse 2.5 parts of DSPE-PEG-Mannose in 50 parts of ultrapure water 2 to obtain liquid B; disperse 15 parts of methotrexate in 30 parts of squalene to obtain liquid C; mix solutions A and B, ultrasonicate for 5 seconds to mix evenly, and then add liquid C, and ultrasonicate together for 2 min to obtain the self-cascading catalytic reactor.
[0030] Figure 2 Characterization of the self-cascading reactor (PECU) prepared with Uri and CAT as stabilizers for Example 2; among them: A. Light microscope image of PECU, B. Particle size of PECU, and C. Zeta potential distribution show that PECU can be successfully prepared by the ultrasonic emulsification method, with good dispersibility and uniform droplet size; D. Catalytic experiments show that compared with the mixed system of the emulsion stabilized by Uri (PEU) and the emulsion stabilized by CAT (PEC), PECU can significantly increase the MSU crystal clearance rate; E. The H2O2 degradation results show that the amount and rate of O2 generated by PECU catalyzing H2O2 are significantly improved, indicating that PECU significantly enhances the cascading catalytic performance.
[0031] Effect Example 1 Using an inverted fluorescence microscope (OLYMPUS U-RFL-T), the intracellular ROS level was measured by the DCFH-DA fluorescent probe. Macrophages were seeded in 96-well plates and incubated overnight. Except for the blank group (n = 6), MSU crystals (0.5 mg / mL) were added to the remaining groups. One hour later, various formulations such as Uri, CAT+Uri, PEU, PEC+PEU, and PECU (20 μg / mL) were added to the culture medium and incubated for another 6 hours. Then, the cells were stained with the DCFH-DA fluorescent probe for 30 min and the nuclei were labeled with DAPI for 2 min. Finally, the intracellular ROS level was detected using an inverted fluorescence microscope. As Figure 3 shown, after macrophages were treated with MSU crystals for 6 hours, oxidative stress was induced to generate a large amount of ROS. However, after treatment with PECU, a lower fluorescence intensity was shown, indicating a strong ROS scavenging ability and effective alleviation of oxidative stress.
[0032] Effect Example 2 Prepare an MSU suspension (10 mg / mL) in PBS, inject 50 μL into the knee joints of mice to establish an acute GA model, and inject an equal volume of PBS into the control mice. Two hours after the MSU crystal injection, ankle swelling and stride gait response in mice were regarded as the criteria for successful model establishment. To evaluate the therapeutic effect, 12 hours after successful modeling, Uri+CAT, PEC+PEU, and PECU (50 μL) were used for treatment. After 72 hours, synovial tissue at the knee joints of each group was collected, and the inflammation was evaluated by H&E staining. As Figure 4 shown in B, obvious inflammatory infiltration was visible in the GA model group. After treatment with the corresponding dosage forms in other groups, the inflammatory responses were alleviated to varying degrees. In particular, the PECU treatment group had a significant anti-inflammatory effect, further confirming the excellent effect of the cascade catalytic reactor in treating GA.
Claims
1. A Pickering emulsion-based cascade catalytic reactor, characterized in that, It consists of uricase and catalase active ingredients, methotrexate, and DSPE-PEG-Mannose.
2. The cascade catalytic reactor according to claim 1, wherein It is composed of the following raw materials in parts by mass: Catalase active ingredient: 500 - 10000 parts Uricase: 500 - 10000 parts Squalene: 20 - 200 parts Methotrexate: 10 - 50 parts DSPE-PEG-Mannose: 1 - 10 parts.
3. The cascade catalytic reactor according to claim 2, wherein The cascade catalytic reactor further includes ultrapure water and a PBS buffer solution with a pH of 7.4 - 8.0 and a concentration of 50 - 160 mM.
4. The cascade catalytic reactor according to any one of claims 1-3, characterized in that The catalase active ingredient is a nanozyme or a protease.
5. The cascade catalytic reactor according to claim 4, wherein The nanozyme is MnO2 nanozyme, Pt nanozyme, Pd nanozyme, or CeO2 nanozyme; the protease is catalase protease.
6. A method for preparing a cascade catalytic reactor according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Disperse the prescribed amount of catalase active ingredient in ultrapure water and stir to obtain liquid A; (2) Disperse the prescribed amount of uricase in ultrapure water to obtain liquid B; (3) Disperse the prescribed amount of DSPE-PEG-Mannose in ultrapure water to obtain liquid C; (4) Disperse the prescribed amount of methotrexate in squalene to obtain liquid D; (5) Mix liquid A, liquid B, and liquid C, ultrasonically mix for 5 seconds, then add liquid D and continue ultrasonic emulsification to obtain a self-cascade catalytic reactor based on Pickering emulsion; Or (a) Disperse uricase and catalase active ingredient in a buffer solution to obtain liquid A; (b) Disperse DSPE-PEG-Mannose in ultrapure water to obtain liquid B; (c) Disperse methotrexate in squalene to obtain liquid C; (d) Mix liquid A and liquid B solutions, ultrasonically mix, then add liquid C and continue ultrasonic treatment to obtain a self-cascade catalytic reactor based on Pickering emulsion.
7. The preparation method according to claim 6, characterized in that, In step (1), the stirring time is 10 - 60 min; in step (5), the ultrasonic time is 1 - 5 min.
8. The preparation method according to claim 6, characterized in that, In step (d), the ultrasonic mixing time is 5 - 10 s; the continued ultrasonic time is 1 - 5 min.
9. Use of a cascade catalytic reactor as described in any one of claims 1 - 6 in the preparation of a drug for treating gouty arthritis.