Construction Of Biomimetic Selfassembled Ceria Nanozyme Drug Based On Special Molecular Linkage For Antiatherosclerosis

Atherosclerosis（AS）,one of the initiating factors of cardiovascular disease,is characterized by chronic inflammation through all stages of the disease.There is growing evidence that reactive oxygen species（ROS）play an important role in the development of AS.Limited by low utilization and toxic side effects,drug-based AS treatment needs to be expanded,and there is an urgent need to develop more effective drugs or strategies to break through the current bottleneck.In recent years,the emergence of nanomaterials with ROS scavenging ability has provided new ideas for the treatment of AS.Among them,ceria nanomaterials have been shown to have significant anti-inflammatory and anti-ROS effects,and have been gradually applied in the therapeutic research of various inflammatory diseases.In view of this,a new strategy based on special molecularly mediated self-assembled ceria nanozyme for anti-atherosclerosis is reported in this paper.Since the interaction between organic molecules and inorganic elements has been a technical bottleneck in the study of nanomaterials,we innovatively screened zoledronic acid（ZOL）as a functional linker molecule and successfully constructed a metal-organic framework-like ceria nanozyme（CZNCs）material with uniform size and rich enzymatic activity;the anti-AS drug probucol（PB）was co-assembled and wrapped by platelet membranes to construct biomimetic ceria nanozymes drug（PCZ@PB NCs）;finally,it was applied to study the ameliorative effect of AS with oxidative stress and inflammatory microenvironment.Both in vitro and in vivo experiments showed that PCZ@PB NCs not only effectively target and accumulate to inflammatory foci of atherosclerosis,but also synergistically regulate the microenvironmental expression levels of ROS and inflammatory factors and inhibit foam cell formation,thus effectively reducing plaque area size and improving the pathological microenvironment for anti-AS.In conclusion,this study provides a versatile assembly technique to obtain biomimetic nanodrugs with multifunctional enzymatic activities and drug delivery capabilities for the treatment of oxidative stress and inflammatory diseases.ObjectiveConstruct a universal drug delivery system based on special molecularly mediated ceria nanozymes,explore its co-assembly and multi-drug loading capabilities and to evaluate its multi-functional enzymatic activity;followed by the construction of biomimetic nanozymes drugs through platelet membrane modification to explore their targeted synergistic pharmacological effects against AS and to provide new ideas and methods for their potential therapeutic functions.A certain theoretical and experimental basis was laid for its further application in biomedical field.Method1.Preparation and characterization of special molecular ligand-mediated self-assembled cerium nanozymes:In order to investigate the feasibility of small molecule-mediated self-assembly of cerium nanozymes,considering the coordination anchoring effect of bisphosphonic acid and imidazole groups on cerium ions,a dul-ligand system of alendronate（AL）and dimethylimidazole（HMIM）was firstly chosen to self-assemble with cerium ions to obtain ceria nanocomposites（CHA）.To simplify the synthesis environment,a special molecule ZOL containing both imidazole and bisphosphonic acid groups in its structure was self-assembled with cerium ions as organic linker molecules to obtain single-ligand ceria nanozymes（CZNCs）.The morphology,physical and chemical properties of CHA and CZNCs were evaluated.In addition,molecular dynamics simulation was used to investigate the self-assembly mechanism of CZNCs in depth.2.Synthesis and characterization of biomimetic ceria nanozyme drugs（PCZ@PB NCs）:To enhance the therapeutic effect of CZNCs,CZNCs and PB were assembled by a one-step method to obtain cerium nanozyme drugs（CZ@PB NCs）.To endow CZ@PB NCs with targeting ability and enhance biocompatibility,platelet membranes were obtained according to the method reported in the literature,and the CZ@PB NCs were encapsulated by ultrasonication to obtain biomimetic cerium nanozyme drugs（PCZ@PB NCs）.The synthesis,physical and chemical properties of CZ@PB NCs and PCZ@PB NCs was characterized.3.In vitro anti-AS activity study of PCZ@PB NCs:Given the important roles played by macrophages,endothelial cells,and vascular smooth muscle cells during AS process,the effect of PCZ@PB NCs on the viability of these three cell types was verified by CCK-8 assay.The ability of PCZ@PB NCs to be taken up by mouse macrophages（Raw 264.7）and inflammatory Raw 264.7 cells,respectively,was evaluated by flow cytometry,as well as the mode of uptake in Raw 264.7 cells.The ability of PCZ@PB NCs to scavenge ROS and the anti-inflammatory effect were verified by fluorescence microscopy,flow cytometry and WB.Oil red O（ORO）staining was used to verify the ability of PCZ@PB NCs to resist oxidized low-density lipoprotein-stimulated Raw 264.7 cellular foaminess.4.In vivo study of the anti-AS effect of PCZ@PB NCs:AS model was established by high-fat feeding Apo E^-/-mice.The distribution in the aorta and major organs was collected by small animal live imager and ICP-MS after tail vein injection of fluorescently labeled PCZ@PB NCs.The anti-AS effect of PCZ@PB NCs was evaluated by ORO staining of longitudinal sections of the aorta as well as frozen sections of the aortic arch,aortic root and brachiocephalic artery in combination with aortic root histology and immunohistochemistry.To examine the biosafety of PCZ@PB NCs,the blood of mice after administration was analyzed for blood biochemical parameters using a fully automated biochemical analyzer,and pathological tests were performed on sections of major organs.Results1.Preparation and characterization of special molecular ligand-mediated self-assembled cerium nanozymes:A clear,transparent and uniform size（10 nm）spherical ceria nanozyme CHA was synthesized by optimizing the synthesis conditions.The loading experiment showed that the dyes Ce6,Cy3 and ICG could participate in the self-assembly of CHA.When CHA was co-assembled with the drug PB,the particle size of the product increased from 10 nm to 20-50 nm but the size was not uniform.The optimization of the synthesis conditions showed that the concentration of cerium nitrate was 5 m M to obtain stable and homogeneous CZNCs spherical nanoparticles with an average size of 10 nm.CZNCs could co-assemble with various dys and drugs to form nanocomposites.Molecular simulations showed that six ZOL molecules were attached around each cerium ion through bisphosphonate-cerium ion and imidazole-cerium ion coordination bonding,forming an ordered backbone structure in turn.Relying on the high affinity of imidazole and bisphosphonic acid groups for different metal ions,ZOL was co-reacted with different metal ions.2.Synthesis and characterization of biomimetic ceria nanozyme drugs（PCZ@PB NCs）:The antioxidant drug molecule PB was co-assembled with CZNCs to obtain CZ@PB NCs.Compared with CZNCs,TEM and AFM showed that CZ@PB NCs had a clear spherical structure,the particle size increased from10 nm to 25 nm,and the hydrated particle size also increased from 15 nm to40 nm.The IR results show that both CZNCs and CZ@PB NCs have typical IR absorption peaks of cerium ion-imidazole groups and Ce-O.In order to endow CZ@PB NCs with active targeting ability,the platelet membrane biomimetic modification was performed to construct PCZ@PB NCs.TEM and uranyl negative staining TEM results showed that the encapsulation of the platelet membrane improved the dispersion of CZ@PB NCs,and a typical"core-shell"structure appeared with an outer membrane thickness of about 3 nm.Compared with CZ@PB NCs,the hydrodynamic size of PCZ@PB NCs is slightly reduced to about 35 nm after membrane encapsulation due to membrane negative charge compression.The valence distribution of PCZ@PB NCs was analyzed by XPS,and the results showed that the distribution of Ce³⁺and Ce⁴⁺on the surface of PCZ@PB NCs were 44.8%and 55.2%,respectively,with little change compared to CZ@PB NCs(Ce³⁺51.7%and Ce⁴⁺48.3%);the detection by dissolved oxygen and ROS showed that CZNCs,CZ@PB NCs and PCZ@PB NCs were able to catalyze H₂O₂ to produce oxygen significantly,and the ROS probe indicated a gradual decrease in H₂O₂ concentration.The inhibition of superoxide anion radical generation by CZ@PB NCs and PCZ@PB NCs for 24 h was verified to be more than 80%using the nitrogen blue tetrazolium assay.In addition,hemoglobin compatibility assay showed that no hemolysis was observed in PCZ@PB NCs at a cerium ion concentration of 200μM.3.In vitro anti-AS activity study of PCZ@PB NCs:CCK-8 experiments demonstrated that cerium ion concentrations above50μM in CZNCs and CZ@PB NCs affected the cell viability of Raw 264.7cells,human umbilical vein endothelial cells（HUVEC）and vascular smooth muscle cells（VSMC）.After biomimetic modification of platelet membranes,PCZ@PB NCs showed no significant effect on the viability of the three cell types even at cerium ion concentrations up to 400μM,and cell safety was improved.Confocal microscopy and flow cytometry analysis showed that PCZ@PB NCs could be efficiently taken up by Raw 264.7 and were more readily internalized by inflammatory Raw 264.7 cells and transported intracellularly via the lysosomal pathway.Fluorescence microscopy,flow cytometry and WB analyses showed that CZNCs,CZ@PB NCs and PCZ@PB NCs could effectively scavenge intracellular ROS and reduce p65protein phosphorylation by levels and downregulated the expression levels of pro-inflammatory factor tumor necrosis factor-α,matrix metalloproteinase-9.ORO staining of intracellular lipid droplets followed by microscopic imaging observed that PCZ@PB NCs reduced ORO staining area most significantly and inhibiting oxidized low-density lipoprotein-mediated foam cell formation.4.In vivo study of the anti-AS effect of PCZ@PB NCs:Fluorescence imaging and ICP-MS quantitative analysis showed that PCZ@PB NCs accumulated more in the aorta than CZ@PB NCs in the AS model mice.In addition,PCZ@PB NCs were most distributed in the liver.Compared with the saline-treated and free PB groups,the PCZNCs and CZ@PB NCs groups had reduced aortic ORO stained areas;ORO stained frozen sections of aortic root,aortic arch and brachiocephalic artery showed that ceruloplasmin inhibited plaque progression;while the necrotic core area was significantly lower in the PCZ@PB NCs group;Masson trichrome staining showed that PCZ@PB NCs treatment group showed higher collagen content around the plaques.In addition,anti-CD68 antibody and anti-MMP-9 antibody staining showed that PCZ@PB NCs treatment effectively reduced macrophage infiltration and MMP-9 expression in plaques.ConclusionIn conclusion,a unique self-assembly strategy of small molecule ligand ZOL with metal ions with versatility was proposed.On this basis,a multifunctional enzyme-active and drug-delivery bionic cerium nanozyme drug PCZ@PB NCs was developed for atherosclerosis treatment.PCZ@PB NCs not only can effectively target to atherosclerotic inflammatory lesions,but also synergistically regulate ROS levels with drugs,inhibit foam cell formation to achieve inhibition of AS process,and exhibit good biocompatibility.On the one hand,it provides a new idea to expand the biomedical application of cerium nanomaterials.On the other hand,in addition to atherosclerosis,this cerium nanoparticle enzyme with ROS scavenging effect has a wide potential for synergistic drug development against other inflammatory diseases.