【作者】 张闯；

【导师】 李正强；

【作者基本信息】 吉林大学 ， 生物物理学， 2022， 博士

【摘要】 酶作为一种高效催化剂,在生物医药、环境及食品工业等领域得到广泛应用。但天然酶的内在缺陷,如易失活、制备成本高、不易长期储存等,大大限制其在实际中的应用。自2007年铁磁纳米粒子首次被报道具有类似过氧化物酶样活性以来,陆续约有300多种纳米材料被发现具有类酶活性,纳米酶越来越为科学家所关注。同天然酶相比,纳米酶具有成本低、易于大规模生产、对恶劣环境具有较高的耐受性、稳定性强且易长期储存等优点,展现出在生物医药领域的应用潜能。金属-有机框架材料（MOFs）是由金属及有机分子配体构筑形成的具有3D网状结构的新型多孔材料。与传统多孔材料相比,MOFs具有明确的结构、高比表面积、可调节的孔径、功能结构可设计性、与生物大分子、聚合物相容等众多优势,基于MOFs本体模拟制造酶性物质的研究蓬勃兴起!旋毛虫病是一种食源性人兽共患寄生虫病,人类因食用含有活的旋毛虫幼虫的肉类而感染,感染旋毛虫病后会出现腹痛、腹泻、恶心呕吐、发热、肌痛、心肌炎、过敏反应、面部水肿和脑炎等症状,严重时可直接致人死亡。因此,如何有效预防和治疗旋毛虫病是科学家们研究的重点。临床上治疗旋毛虫病主要是抗蠕虫咪唑类药物,由于咪唑类药物可溶性差且长期服用会产生一定副作用,导致生物体内抗氧化系统紊乱,寻找新的治疗手段和方法尤为重要。有学者研究发现过氧化物酶系统在体内对旋毛虫杀伤具有重要的生物学意义。同时,旋毛虫利用环境及宿主体内的葡萄糖,通过糖酵解等方式为自身分化及成长提供所需的能量。利用葡萄糖氧化酶可将葡萄糖分解成H₂O₂,减少旋毛虫体内能量供给,又为过氧化物酶提供充足的H₂O₂原料。因此,酶学方法治疗旋毛虫病将是一个新的研究思路。综合级联反应的优势,将过氧化物酶和葡萄糖氧化酶级联,有助于增强多酶体系的反应活性,构建构效优良双酶体系对旋毛虫杀伤具有重要意义。但两类酶的简单混合,不能形成稳定的结合体,继而降低级联反应的活性,酶的固定化将有效解决这一不足。我们前期研究发现MOF能吸附蛋白及染料,并具有过氧化物酶活性。因此,选用具有过氧化物酶活性的MOF作为固定化载体,兼具载体及催化能力双重优势。酶可通过静电吸附作用固定在MOF载体上,但易受p H的影响从MOF上脱落,采用酶共价固定的方式将避免该现象的发生。PEG作为一种高分子聚合物,具有官能团可修饰性及良好的生物相容性、安全性。选择PEG作为中间体,将MOF与酶连接形成双酶体系,既增加体系的稳定性,又弥补MOF在水溶液中分散性差的缺点。基于以上启示,本工作以具有类过氧化物酶活性的MOF-525（Fe）（简称MOF-Fe）为载体,采用双功能化的PEG（NH2-PEG-COOH）将MOF-Fe与GOx偶联,开发一种新的双酶体系,简称MPG（Fe）,并对MPG（Fe）抗旋毛虫的作用及分子机制进行了研究。1、成功合成MPG（Fe）双酶体系并对其结构进行表征。SEM&TEM显示MPG（Fe）呈约30 nm大小的圆球形;UV-Vis显示铁离子成功与卟啉配位、XRD显示合成的MPG（Fe）晶体结构是正确的,完整的;XPS数据显示,PEG已成功与MOF-Fe上的Zr6簇连接;Zeta-电位数据显示MOF-Fe经PEG、GOx修饰后,在PBS介质中的分散性逐渐增强,有助于该双酶体系在动物体内的应用。MOF-Fe吸附和负载GOx的稳定性实验,结果显示,负载GOx合成的MPG（Fe）不受p H变化影响,稳定性强于吸附合成的MOF-Fe@GOx。2、分析MPG（Fe）双酶体系的类过氧化物酶活性和葡萄糖氧化酶活性。结果表明,当反应体系中含有葡萄糖时,MPG（Fe）能将葡萄糖直接氧化成·OH,使ABTS转变成ABTS+。ESR实验中,使用DMPO捕获·OH,出现典型的DMPO/·OH 4倍特征峰,具有1:2:2:1的相对强度;荧光光谱法也证明反应过程中生成·OH。对MPG（Fe）过氧化物酶活性分析发现,在p H 4.0时,该双酶体系具有最适催化活力;稳态动力学研究发现MPG（Fe）对H₂O₂的KM值为5.81 m M,低于已报道的其它过氧化物酶纳米酶,对H₂O₂的亲和力高于已报道的其它纳米酶,有利于催化H₂O₂生成·OH。在MPG（Fe）的GOx活性研究中发现,负载的GOx依然保持酶活性,能催化葡萄糖氧化生成H₂O₂。催化反应过程中产生的葡萄糖醛酸能降低反应体系的p H,在MPG（Fe）浓度200μg/m L反应24 h后,p H由7.4下降至5.5左右,p H的下降为过氧化物酶催化反应提供有力条件。3、体外MPG（Fe）双酶体系抗旋毛虫实验中发现,MPG（Fe）具有明显抗旋毛虫成虫和新生幼虫的作用,减少旋毛虫成虫及新生幼虫在宿主体内的数量,进而降低旋毛虫病感染对机体的损伤程度。浓度为100μg/m L的MPG（Fe）作用8 h后,成虫死亡率达到70%,12 h后死亡率达100%。新生幼虫经浓度为5μg/m L的MPG（Fe）作用12 h后,死亡率达95%以上;当给药浓度为100μg/m L时,新生幼虫在2 h内完全死亡。细胞实验显示,MPG（Fe）影响猪小肠上皮细胞的活力,MOF-Fe对细胞活力无明显影响,MPG（Fe）导致细胞活性降低源于偶联GOx消耗培养基中的葡萄糖,细胞因无葡萄糖而活力降低。4、体内实验表明,MPG（Fe）双酶体系具备抗旋毛虫成虫的作用。经高剂量组（15 mg/kg）给药后,成虫生存率仅为3.53%。通过对小鼠血细胞分析、流式细胞术分析免疫细胞、脾组织免疫荧光染色及心、肝、脾、肺、肾组织病理切片评估MPG（Fe）生物安全性,结果表明,小鼠经短期和长期两种注射方式后,低浓度（2.5 mg/kg）、中浓度（7.5 mg/kg）、高浓度（15 mg/kg）实验组与PBS组无明显区别,表明MPG（Fe）具有良好的生物安全性,是一种既安全又有效的纳米双酶体系。综上,MPG（Fe）双酶体系同时具有过氧化物酶活性和葡萄糖氧化酶活性,有较好的稳定性,可以有效降低旋毛虫感染部位周围葡萄糖水平,减少旋毛虫能量摄入。同时,产生氧化性更强的·OH增强抗旋毛虫效果,且在体内具有良好的生物安全性,是一种既安全又有效的双酶体系。本工作为纳米酶与天然酶双酶体系在人兽共患寄生虫病领域应用提供新思路,也为旋毛虫病提供一个新的治疗手段。更多还原

【Abstract】 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.更多还原