【作者】 张帅；

【导师】 孙健；

【作者基本信息】 吉林大学 ， 内科学， 2023， 博士

【摘要】 研究背景:活性氧（reactive oxygen species,ROS）作为信号分子参与多种信号通路的调控,在调节细胞生长、分化和凋亡方面发挥着重要的生理作用。过量的ROS可诱发基因的突变、蛋白质变性和脂质过氧化,导致细胞损伤及坏死。内源性损伤相关分子模式（damage-associated molecular patterns,DAMPs）通过激活一系列信号转导通路,进而介导机体炎症反应,使得损伤组织处于持续的氧化应激和炎症微环境中。过量高活性的ROS在动脉粥样硬化（atherosclerosis,AS）和缺血灌注损伤（ischemia-reperfusion injury,IRI）的病理过程中发挥着重要的作用。因此,抑制ROS生成并阻断炎症反应成为AS和IRI治疗策略之一。由于稳定性差、递送效率低以及缺乏组织特异性,传统抗氧化剂可能对特定器官的生物利用度较低而无法发挥理想治疗作用。因此,亟需开发一种具有靶向特异性、高生物利用度的新型抗氧化剂。纳米医学的迅速发展极大地促进生物医药技术的革新,尤其在抗氧化治疗领域取得重大进展。基于纳米材料构筑新型抗氧化剂已经成为氧化应激相关疾病治疗的研究热点之一。纳米材料可以作为药物优良载体,极大地改善天然抗氧化分子的药代动力学,并保护其清除ROS活性免受组织微环境的影响。纳米酶是近年来发现的具有类似生物酶的酶促反应动力学特性的功能纳米材料。与天然生物酶相比,纳米酶展现了高效ROS清除能力和更高的抗氧化稳定性,未来有望成为抗氧化及抗炎治疗的新型纳米药物。因此,本研究设计与构建了两种具有炎性微环境靶向性和多种模拟酶活性的新型纳米药物,并通过体内外实验分别探究其在AS斑块和肝脏IRI中的治疗效果及作用机制。第一部分CeO₂@CD/SIM-Ab纳米酶平台的构建、表征及性质测定目的:基于易损斑块微环境,构建一种具有衰老泡沫细胞靶向功能的纳米酶平台,并对其结构和性质进行表征。方法:通过一步水热法制备β-环糊精（β-cyclodextrin,β-CD）功能化的二氧化铈（cerium oxide,CeO₂）纳米棒（C@CD）。利用主客体相互作用和物理吸附作用,实现药物辛伐他汀（Simvastatin,SIM）的负载。利用聚乙烯亚胺对C@CD表面进行氨基功能化修饰,进一步利用SMCC法将CD9抗体偶联至材料表面,最后制备得到CeO₂@CD/SIM-Ab纳米酶平台（C@CDS-Ab）。利用多种表征方法对C@CDS-Ab的形貌、结构、孔径分布、元素组成及价态等进行表征。在体外水平对C@CDS-Ab的超氧化物歧化酶（superoxide dismutase,SOD）和过氧化氢酶（catalase,CAT）模拟酶活性进行测定。利用紫外吸收光谱对C@CDS-Ab的药物负载能力进行评价。结果:我们首先制备了具有多孔结构的β-CD功能化的CeO₂纳米棒（C@CD）,其平均孔径为9.487 nm,平均水合粒径50.7 nm,zeta电位值为-20.9±0.56 m V。通过进一步药物负载及抗体功能化,最终成功制备C@CDS-Ab纳米酶平台。C@CDS-Ab的平均水合粒径约为190 nm,zeta电位值为6.55±1.36 m V,且具有良好稳定性。C@CDS-Ab载药能力和载药效率分别为20.85%和26.34%。体外实验证实了C@CDS-Ab可有效清除O₂^·-和H₂O₂等氧自由基。结论:C@CDS-Ab纳米酶平台不仅是优良的药物载体,能够实现SIM的高效负载和靶向递送;并且C@CDS-Ab纳米酶平台具有优异的SOD和CAT模拟酶活性,可以协同SIM发挥抗氧化与抗炎活性。第二部分CeO₂@CD/SIM-Ab纳米酶平台的抗动脉粥样硬化作用研究目的:在细胞和动物水平,探究C@CDS-Ab纳米酶平台的抗AS作用。方法:首先,利用氧化型低密度脂蛋白（oxidizes low density lipoprotein,Ox-LDL）诱导RAW264.7细胞建立衰老泡沫巨噬细胞模型。利用荧光素标记C@CDS-Ab,评价其对衰老泡沫巨噬细胞的靶向能力。利用ROS荧光探针、油红O染色和酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）等方法,探究C@CDS-Ab的抗氧化和抗炎作用,及其对巨噬细胞泡沫化的影响。随后,通过荧光成像和CT成像技术,评价C@CDS-Ab在小鼠主动脉炎性斑块中靶向富集能力。最后,通过给予载脂蛋白E基因敲除(apolipoprotein E gene-deficient,Apo E^-/-)小鼠高脂饮食8周,建立AS小鼠模型。随机挑选40只小鼠入组实验,随机分成4组:（1）模型组;（2）C@CD-P治疗组;（3）C@CDS-P治疗组;（4）C@CDS-Ab治疗组。通过主动脉油红O染色和主动脉根部切片组织化学染色等技术评价C@CDS-Ab的抗AS作用。结果:细胞实验中,C@CDS-Ab能够被衰老泡沫巨噬细胞摄取,且呈现时间依赖性。细胞荧光染色结果显示C@CDS-Ab能清除细胞内ROS。细胞油红O染色表明C@CDS-Ab能够抑制巨噬细胞泡沫化,ELISA显示C@CDS-Ab显著减少了巨噬细胞分泌TNF-α和IL-6。体内实验中,C@CDS-Ab能够靶向富集在炎性斑块中,并且通过负载碘佛醇,可以实现炎性斑块的CT成像。在治疗方面,与模型组相比,C@CDS-Ab治疗组主动脉斑块面积明显减少（P<0.001）,平均斑块面积占比降低至9.67%,且斑块内具有较少的脂质坏死核心和较高的胶原蛋白含量。免疫组织化学染色分析显示,与模型组主动脉斑块相比,C@CDS-Ab治疗组斑块内巨噬细胞含量及MMP-9表达量明显减少（P分别<0.001和<0.05）。结论:（1）C@CDS-Ab纳米酶平台能够特异性靶向衰老泡沫巨噬细胞,并在体内实现在炎性斑块中的靶向富集。（2）C@CDS-Ab能够清除细胞内ROS,抑制巨噬细胞泡沫化,减少衰老泡沫细胞形成,有效地减少促炎因子的分泌。（3）C@CDS-Ab能够有效地减少斑块的形成,延缓AS的进展。通过提高斑块内胶原蛋白含量,减少巨噬细胞浸润,降低MMP-9的表达水平,减轻斑块微环境中炎症反应,从而增加了斑块的稳定性。第三部分CeO₂@MnO₂复合纳米酶对肝脏缺血再灌注损伤的保护作用目的:基于肝脏IRI炎性微环境,构建一种具有Kupffer细胞（KCs）被动靶向功能的CeO₂@MnO₂复合纳米酶（CM NCs）,探究其对肝脏IRI的保护作用。方法:通过生物矿化法制备血清白蛋白（bovine serum albumin,BSA）功能化的CM NCs。首先,对CM NCs的形貌、结构、元素组成及价态等进行表征。并在体外水平对CM NCs的SOD、CAT模拟酶活性及清除羟基自由基（hydroxyl free radicals,?OH）能力进行测定。利用ROS荧光探针和细胞活力检测等方法,探究CM NCs对细胞氧化应激损伤的保护作用。随后在体内水平验证CM NCs对肝脏组织及KCs的被动靶向能力。最后,在肝脏部分IRI小鼠模型中,通过血清肝功酶学指标检测、伊红-苏木素染色、二氢乙啶荧光染色、肝脏组织匀浆中的丙二醛（malondialdehyde,MDA）及炎性细胞因子水平检测等方法,探究CM NCs在缺血再灌注过程中对肝脏的保护作用。结果:CM NCs具有尺寸小、单分散的特点,直径约为2 nm,平均水合粒径23.12 nm,zeta电位值为-18.67 m V。体外水平证实了CM NCs可有效清除O₂^·-、H₂O₂和?OH等氧自由基。细胞实验中,CM NCs能够有效被RAW264.7细胞摄取,且呈现时间依赖性。CM NCs能降低H₂O₂诱导Hep G2细胞内ROS水平,有效减轻细胞氧化应激损伤。体内实验中,经尾静脉给药6 h后,CM NCs在肝脏中富集量达到峰值;第14天,肝脏组织中大部分CM NCs被代谢清除。生物透射电子显微镜进一步证实了CM NCs能够被肝脏KCs有效摄取。在肝脏部分IRI小鼠模型中,与IRI组相比较,CM NCs预处理组小鼠肝组织损伤程度较轻,少量的肝细胞胞质空泡化,肝组织匀浆中MDA水平显著降低（P<0.05）。此外,与IRI组相比较,CM NCs预处理组小鼠肝组织匀浆中TNF-α、IFN-γ和MPO等炎性因子的含量明显降低（P<0.01）。结论:（1）我们成功构建了小尺寸的CeO₂@MnO₂复合纳米酶（CM NCs）,其具有高生物相容性、肝脏KCs被动靶向性和短期代谢清除等优点。（2）CM NCs具有SOD、CAT等多种模拟酶活性,可协同清除O₂^·-、H₂O₂和?OH等氧自由基。（3）CM NCs能够有效清除组织再灌注过程中产生过量的ROS,降低脂质过氧化水平,维持氧化还原平衡状态,减轻肝脏组织氧化应激损伤。（4）CM NCs通过降低活化KCs来源的促炎症细胞因子的产生,减少微环境内中性粒细胞的募集,减轻损伤组织中的炎症反应。更多还原

【Abstract】 Background:As signal molecules,reactive oxygen species（ROS）participate in the regulation of a variety of signal pathways and play an important physiological role in regulating cell growth,differentiation and apoptosis.Excess ROS can induce mutations in genes,protein denaturation and lipid peroxidation,leading to cellular damage and necrosis.Endogenous damage-associated molecular patterns（DAMPs）mediate the inflammatory response by activating a series of signal transduction pathways,which in turn leave injured tissues in a continuous oxidative stress and inflammatory microenvironment.Abnormal expression of ROS plays an important role in the pathological process of atherosclerosis（AS）and ischemic-perfusion injury（IRI）.Therefore,inhibition of ROS production and blocking the inflammatory response has become one of the therapeutic strategies for AS and IRI.Due to poor stability,low delivery efficiency,and lack of tissue specificity,conventional antioxidants may be less bioavailable to specific organs for their therapeutic effects.Therefore,there is an urgent need to develop a novel antioxidant with tissue specificity and high bioavailability.The rapid development of nanomedicine has greatly promoted the innovation of biomedical technology,especially in the field of antioxidant therapy.The construction of novel antioxidants based on nanomaterials has become one of the research hotspots for the treatment of oxidative stress-related diseases.Nanomaterials can be used as excellent drug carriers to improve the pharmacokinetics of natural antioxidant molecules and protect their ROS scavenging activity from the tissue microenvironment.Compared with natural biological enzymes,nanoenzymes exhibit efficient ROS`scavenging ability and higher antioxidant stability,and are expected to be novel nanomedicines for antioxidant and anti-inflammatory therapy in the future.Therefore,in this study,two novel nanodrugs with inflammatory microenvironment targeting and multiple mimic enzyme activities were constructed,and their therapeutic effects and mechanisms in AS plaques and hepatic IRI were investigated in vivo and in vitro.Part 1.Construction and characterization of CeO₂@CD/SIM-Ab nanoenzyme platformObjective:Construction and characterization of a nanoenzyme platform with senescent foam macrophages targeting based on vulnerable plaque microenvironment.Methods:Cerium oxide nanorods functionalized byβ-cyclodextrin（β-CD）were prepared by one-step hydrothermal method.The loading of simvastatin（SIM）was realized by host-guest interaction and physical adsorption.Polyethyleneimine was used to functionalize the surface of C@CD to obtain amino functional group.The CD9antibody was coupled to the material surface using the SMCC method,and finally CeO₂@CD/SIM-Ab nanoenzyme platform（C@CDS-Ab）was prepared.Various characterization methods were used to characterize the morphology,structure,pore size distribution,elemental composition and valence of C@CDS-Ab.The SOD and CAT mimic enzyme activities of C@CDS-Ab were determined in vitro.The drug loading capacity of C@CDS-Ab was evaluated by ultraviolet absorption spectrum.Results:Firstly,β-CD functionalized CeO₂ nanorods（C@CD）with porous structure were prepared.The average pore size is 9.487 nm and the average hydrated particle size is 50.7 nm.Through further drug loading and antibody functionalization,the C@CDS-Ab nano-enzyme platform was successfully prepared.The average hydrated particle size of C@CDS-Ab is about 190 nm and the zeta potential is 6.55±1.36 m V,and it has good stability.The drug loading capacity and drug encapsulation efficiency of C@CDS-Ab were 20.85%and 26.34%,respectively.In vitro,it was confirmed that C@CDS-Ab could scavenge oxygen free radicals such as O₂^·-and H₂O₂.Conclusions:The C@CDS-Ab nanoenzyme platform is not only an excellent drug carrier for efficient loading and targeted delivery of SIM,but also has excellent SOD and CAT mimetic enzymatic activities,which can synergize with SIM for antioxidant and anti-inflammatory activities.Part 2.Anti-atherosclerotic effect study of CeO₂@CD/SIM-Ab nanoenzyme platformObjective:Explore the anti-atherosclerotic effects of C@CDS-Ab nanoenzyme platform at cellular and animal models.Methods:The oxidized low density lipoprotein（Ox-LDL）-treated macrophages（senescent foam macrophage formation）were selected as in vitro models.Fluorescein-labeled C@CDS-Ab was used to evaluate its targeting ability on senescent foam macrophages.ROS fluorescence probe,oil red O staining and enzyme linked immunosorbent assay（ELISA）were used to explore the antioxidant and anti-inflammatory effects of C@CDS-Ab and its effect on cell foaming.Then,the targeting ability of C@CDS-Ab for plaques in the aortas of mice was evaluated by fluorescence imaging and CT imaging.Finally,a mouse model of AS was estabilished by feeding apolipoprotein E gene-deficient(Apo E^-/-)mice with a high-fat diet for 8 weeks.Forty mice were randomly divided into 4 groups:（1）model group;（2）C@CD-P treated group;（3）C@CDS-P treated group;（4）C@CDS-Ab treated group.The anti-atherosclerotic effects of C@CDS-Ab was evaluated by aortic oil red O staining and histochemical staining of aortic root sections.Results:In cellular assays,C@CDS-Ab was taken up by senescent foam macrophages in a time-dependent manner.Cytofluorimetric staining showed that C@CDS-Ab could scavenge intracellular ROS.Oil red O staining showed that C@CDS-Ab could inhibit the foaming of macrophages,and ELISA confirmed that C@CDS-Ab could effectively reduce the secretion of TNF-αand IL-6 by macrophages.In vivo experiments showed that C@CDS-Ab was able to target toward inflammatory plaques and could be used for CT imaging of inflammatory plaques via loading ioversol.In terms of treatment,compared with the model group,the aortic plaque area in the C@CDS-Ab treated group decreased significantly（P<0.001）,the average plaque area decreased to 9.67%,and the plaque had less lipid necrotic core and higher collagen content.Further immunohistochemical analysis showed that the content of macrophages and the expression of MMP-9 in C@CDS-Ab treated group were significantly lower than those in model group（P<0.001 and P<0.05,respectively）.Conclusions:（1）The C@CDS-Ab nanoplatform is able to specifically target senescent foam macrophages and achieve targeted enrichment in inflammatory plaques in vivo.（2）C@CDS-Ab can scavenge intracellular ROS,inhibit macrophage-to-foam cell transformation,reduce the formation of senescent foam macrophages,and effectively reduce the secretion of pro-inflammatory cytokines.（3）C@CDS-Ab can effectively reduce plaque formation and delay the progression of AS.C@CDS-Ab can increase collagen content within the plaque,reduce macrophage infiltration,decrease MMP-9 expression levels,and attenuate inflammatory responses in the plaque microenvironment,thus increasing the stability of plaque.Part 3.Protective effect of CeO₂@MnO₂ composite nanoenzymes on hepatic ischemia-reperfusion injuryObjective:Based on the inflammatory microenvironment of hepatic IRI,CeO₂@MnO₂ composite nanoenzymes（CM NCs）with passive targeting for Kupffer cells（KCs）were constructed to investigate their protective effect against hepatic IRI.Methods:Serum albumin（BSA）-functionalized CM NCs were prepared by a biomineralization method.Firstly,the morphology,structure,element composition and valence of CM NCs were characterized.The activities of SOD and CAT mimic enzymes and the ability of scavenging hydroxyl free radicals（?OH）of CM NCs were measured in vitro.The protective effects of CM NCs against cellular oxidative stress damage were investigated using ROS fluorescent probes and cell viability assays.Then the passive targeting ability of CM NCs in liver tissue and KCs was verified in vivo.Finally,in the mouse model of partial hepatic IRI,the protective effect of CM NCs on liver during ischemia-reperfusion was investigated by detection of hepatic function markers,eosin-hematoxylin staining,dihydroethidium fluorescence staining,and detection of malondialdehyde（MDA）and inflammatory cytokines in hepatic tissue homogenates.Results:CM NCs has the characteristics of small size and monodisperse.Their average diameter were about 2 nm and average hydrated particle size is 23.12 nm.The zeta potential of CM NCs was-18.67 m V.In vitro,it was confirmed that CM NCs could scavenge oxygen free radicals such as O₂^·-,H₂O₂ and?OH.In cellular assays,CM NCs were effectively taken up by RAW264.7 cells in a time-dependent manner.CM NCs can reduce the level of ROS in Hep G2 cells induced by H₂O₂,and effectively attenuate the damage caused by oxidative stress.In vivo experiment,the concentration of CM NCs in liver reached the peak after 6 hours of administration through tail vein,and most of CM NCs in liver tissue was metabolized and cleared on the 14th day.Biotransmission electron microscopy further revealed that CM NCs could be effectively taken up by hepatic KCs.In the mouse model of partial hepatic IRI,the CM NCs pretreated group showed a small amount of hepatocyte cytoplasmic vacuolization,and the level of MDA in hepatic tissue homogenates were significantly decreased compared with IRI group（P<0.05）.In addition,the levels of inflammatory cytokines such as TNF-α,IFN-γand MPO in hepatic tissue homogenates of mice pretreated with CM NCs were significantly lower than those of IRI group（P<0.01）.Conclusions:（1）We successfully constructed CeO₂@MnO₂ composite nanoenzymes（CM NCs）with the advantages of small size,high biocompatibility,passive targeting of liver KCs and short-term metabolic clearance.（2）CM NCs have various mimetic enzymatic activities such as SOD and CAT,which can synergistically scavenge oxygen radicals such as O₂^·-,H₂O₂and?OH.（3）CM NCs can effectively eliminate the excessive ROS produced during tissue reperfusion,reduce the level of lipid peroxidation,maintain the balance of redox and reduce the oxidative stress injury of liver tissue.（4）CM NCs can reduce the production of pro-inflammatory cytokines derived from activated KCs,reduce the recruitment of neutrophils within the microenvironment,and reduce the inflammatory response in the injured hepatic tissue.更多还原