Study On Construction Of MPG(Fe) Dual Enzyme System And Its Effect Of Killing Trichinella Spiralis

As an efficient catalyst,enzymes are widely used in biomedical,environmental and food industries.However,the inherent defects of natural enzymes,such as easy deactivation,high preparation cost,and difficult long-term storage,greatly limit their application in practice.Since the first report of ferromagnetic nanoparticles with peroxidase-like activity in 2007,more than 300 nanomaterials have been found to have enzyme-like activities,and nanozymes are gaining more and more attention from scientists.Compared with natural enzymes,nanozymes have shown potential for biomedical applications because of their low cost,easy mass production,high tolerance to harsh environments,and high stability and long-term storage.Metal-organic framework materials（MOFs）are novel porous materials with 3D mesh structure formed by metal and organic molecular ligand constructs.Compared with conventional porous materials,MOFs have many advantages of well-defined structure,high specific surface area,adjustable pore size,designability of functional structure,and compatibility with biomolecules and polymers,etc.Research on the fabrication of enzymatic substances based on MOFs ontology simulation is booming!Trichinosis is a food-borne zoonotic parasitic disease that infects humans by eating meat containing live Trichinella larvae.Infection with trichinosis can cause abdominal pain,diarrhea,nausea and vomiting,fever,myalgia,myocarditis,allergic reactions,facial edema and encephalitis,and even direct death.Therefore,how to effectively prevent and treat trichinosis is the focus of scientists’ research.The clinical treatment of trichinosis is mainly based on anthelmintic drugs,but due to the poor solubility of imidazole drugs and the side effects of long-term use,the antioxidant system in the organism is disrupted,so it is important to find new treatments and methods.Studies have shown that the peroxidase system is biologically important for Trichinella killing in vivo.At the same time,Trichinella can use glucose from the environment and the host to provide energy for its differentiation and growth by means of glycolysis.Glucose oxidase can be used to break down glucose into H₂O₂,reducing the energy supply in Trichinella and providing sufficient H₂O₂ raw material for peroxidase.Peroxidase and glucose oxidase tandem initiation of cascade catalysis offers the possibility to seek killing Trichinella in vivo.However,the simple mixture of these two types of enzymes is randomly distributed in vivo,thus increasing the time for the glucose oxidase product H₂O₂ to activate peroxidase catalysis and decreasing the reaction system H₂O₂ concentration and subsequently the activity of the cascade reaction.Immobilization of the two types of enzymes on suitable carriers can help to compensate for the lack of simple mixing,so it is important to construct a conformationally excellent dual enzyme system.In this work,MOF-525（Fe）（MOF-Fe）,which has the advantages of both peroxidase activity and immobilized carrier,was used as the core,and a biofunctionalized PEG（NH2-PEG-COOH） was used to couple MOF-Fe with GOx to synthesize a new dual enzyme system complex,referred to as MPG（Fe）,and the antitrichinosis effect and molecular mechanism of MPG（Fe）were investigated.1.MPG（Fe） dual enzyme system was successfully synthesized and its structure was characterized.SEM&TEM showed that MPG（Fe）was in the shape of a round sphere of about 30 nm size;UV-Vis showed that Fe ions were successfully coordinated with porphyrins,XRD showed that the crystal structure of the synthesized MPG（Fe）was correct and intact;XPS data showed that PEG had been successfully attached to the Zr6 cluster;Zeta-potential data showed that MOF-Fe was modified by PEG and GOx,and the dispersion in PBS medium was gradually enhanced,which will help the application of this complex in organisms.Meanwhile,the stability of MOF-Fe adsorbed and loaded with GOx was explored,and the results showed that the synthesized MPG（Fe）loaded with GOx was not affected by p H change and was more stable than the adsorbed synthesized MOF-Fe@GOx.2.MPG（Fe） dual enzyme system was analyzed for peroxidase-like activity and glucose oxidase activity.The results showed that MPG（Fe） could directly oxidize glucose to ·OH when the reaction system contained glucose,converting ABTS to ABTS+.ESR experiments,using DMPO to capture ·OH,showed a typical DMPO/·OH4-fold characteristic peak with a 1:2:2:1 relative intensity;fluorescence spectroscopy also demonstrated the generation of ·OH during the reaction;analysis of MPG（Fe） peroxidase activity analysis revealed that the complex had the optimum catalytic activity at p H 4.0;the steady-state kinetic study found that the KM value of MPG（Fe） for H₂O₂ was 5.81 m M,which was lower than that of other reported peroxidase nanozymes,and the affinity for H₂O₂ was higher than that of other reported nanozymes,which was favorable to catalyze the generation of ·OH from H₂O₂.In the MPG（Fe）GOx activity study,it was found that the loaded GOx still maintained enzymatic activity and could catalyze glucose oxidation and H₂O₂ generation.Meanwhile,the glucuronic acid produced during the catalytic reaction could lower the p H of the reaction system.The p H decreased from 7.4 to 5.5 after 24 h of reaction at MPG（Fe）concentration of 200 μg/m L,and the decrease of p H provided strong conditions for the peroxidase-catalyzed reaction.3.In the investigation of MPG（Fe）dual enzyme system against Trichinella in vitro,it was found that MPG（Fe）had a significant effect against AD and NBL in vitro,reducing the number of AD and NBL in the host and thus reducing the degree of damage to the organism by Trichinella infection.The mortality rate of AD reached 70% after 8h and 100% after 12 h of MPG（Fe） at a concentration of 100 μg/m L.The mortality rate of NBL reached more than 95% after 12 h of MPG（Fe）at a concentration of 5 μg/m L,and the NBL died completely within 2 h when administered at a concentration of 100μg/m L.Cellular assays showed that MPG（Fe） affected the viability of porcine small intestinal epithelial cells,while MOF-Fe had no significant effect on cell viability.The decrease in cell activity due to MPG（Fe）resulted from the consumption of glucose in the medium by coupled GOx,and the cells were less viable due to the absence of glucose.4.MPG（Fe） dual enzyme system can still effectively play the role of killing AD in vivo,and the survival rate of AD was only 3.53% after administration by high dose group（15 mg/kg）.The biological safety of MPG（Fe）was assessed by blood cell analysis,flow cytometry analysis of immune cells,immunofluorescence staining of spleen tissue and histopathological sections of heart,liver,spleen,lung and kidney of mice.The results showed that MPG（Fe）has good biosafety and is a safe and effective dual enzyme system.In conclusion,MPG（Fe） has both peroxidase activity and glucose oxidase activity,and has good stability,which can effectively reduce the glucose level around the trichinella infection site and reduce trichinella energy intake.At the same time,it produces stronger oxidative ·OH to enhance the killing trichinella effect and has good biosafety in vivo,which is a safe and effective dual enzyme system.This work provides a new idea for the application of the dual enzyme system of nanozymes and natural enzymes in the field of zoonotic parasitic diseases,and also provides a new treatment for trichinosis.