Construction Of Artemisinin And Procyanidins-loaded Biomimetic Nanosystem And Its Anti-atherosclerosis Study

Atherosclerosis is a slow,multifactorial arterial wall disease involved by multiple vessels.The early clinical treatment focus on drug and diet intervention and the late clinical treatment concentrates on surgical intervention,while both of them have certain limitations and disadvantages.It is necessary to construct effective therapeutic strategies to intervene the occurrence and development of atherosclerotic lesions targeted on specific cellular and molecular mechanisms.The development of atherosclerosis is closely related to macrophages,and the inhibition of lipid influx and the promotion of cholesterol efflux in macrophages could delay the progression of atherosclerosis.Natural compounds have great potential in the prevention and treatment of atherosclerosis.Studies have shown that artemisinin（ART）and procyanidins（PC）have been reported in the treatment of atherosclerosis with ART reduce inflammation to inhibit lipid influx and PC enhance autophagy to promote cholesterol efflux.The combination of ART and PC would be an effective regimen for the clinical treatment of atherosclerosis.I n this work,the combinational effects and mechanism of ART and PC on lipid influx and cholesterol efflux were explored by using network pharmacology,molecular docking and related biochemical experiments.However,ART is a liposoluble compound and PC is a water-soluble compound,which limit their simultaneous delivery to atherosclerotic plaque sites.In order to overcome the pharmacokinetic limitation of free drugs and achieve simultaneous drug delivery,we constructed a biomimetic nanosystem to deliver AR T and PC to the plaque site,and then systematically investigated its anti-atherosclerosis effect and mechanism.This work hope to provide new strategies for the application and functional design of natural compounds in atherosclerosis by taking the combin ation of ART and PC as an example.Main research contents are as follows:（1）Investigate the combinational effect of ART and PC on lipid influx and cholesterol efflux of macrophages.In this part,network pharmacology was used to analyze the relationship between ART,PC and atherosclerosis,and results indicated that ART and PC showed the characteristics of multi-target,multi-pathway and multi-biological function in the treatment of atherosclerosis,mainly involves the IL-17 signaling pathways,NOD receptors signaling pathways,TNF signaling pathways,autophagy and AMPK signaling pathway.The interaction between ART,PC and potential targets were preliminarily verified by molecular docking.A nd results showed that ART and PC interacted with NF-κB/NLRP3 pathway and inflammatory factors,as well as AMPK/mTOR/autophagy pathway and cholesterol efflux related proteins.Anti-atherosclerosis effects of ART and PC were further studied by ROS scavenging assay,NO scavenging assay,lipid uptake assay and oil Red O staining assay.And results shown that the combination of ART and PC has good inhibitory effect on the production of ROS and NO,as well as reduce the uptake and deposition of oxLDL in macrophages,leading to inhibit the formation of foam cells.Western bl otting and ELISA assay were used to analyze the main molecular mechanisms of ART and PC.Results showed that the combination of ART and PC can inhibit lipid influx of macrophages by regulating the RONS/NF-κB/NLRP3/inflammation pathway and effectively promote cholesterol efflux of macrophages by enhancing AMPK/mTOR/autophagy pathway.In conclusion,the combination of ART and PC is expected to be used in the treatment of atherosclerosis via simultaneously modulating lipid influx and cholesterol efflux.（2）Construct and characterize of biomimetic nanosystems HA-M@PB@（PC+ART）NPs for targeted delivery of ART and PC.In order to overcome the pharmacokinetic limitation of free drugs and further improve the anti-atherosclerosis efficacy of ART and PC,we constructed a biomimetic nanosystem to deliver ART and PC to the site of atherosclerotic plaque:hollow mesoporous prussian blue nanoparticles（PB NPs）were prepared as the carrier to realize the efficient loading of ART and PC to form PB@（PC+ART）NPs.Then,hybrid membrane formed by red blood cell membrane（RBCm）and macrophage membrane（M?m）were camouflaged on the surf ace of PB@（PC+ART）NPs to form M@PB@（PC+ART）NPs with bionic properties,which could help nanoparticles to prolong the circulation time in the blood and realize the effective accumulation of atherosclerotic plaque sites.Furthermore,hyaluronic acid（HA）w as inserted into the surface of the bionic hybrid membrane to prepare HA-M@PB@（PC+ART）NPs with specific targeting properties for inflammatory macrophages in plaques area.The preparation process and physicochemical characteristics of HA-M@PB@（PC+ART）NPs were characterized by Transmission electron microscopy（TEM）,UV-vis spectrum（UV-vis）,Fourier transform infrared spectroscopy（FTIR）,X-ray photoelectron spectroscopy（XPS）,X-ray diffraction spectroscopy（XRD）,and dynamic light scattering analysis（DLS）.And above results indicated that the biomimetic nanosystem HA-M@PB@（PC+ART）NPs were constructed,which have coated by biomimetic membrane with high-efficiency loading of ART and PC and particle size about 150 nm.Results of Forster resonance energy tra nsfer（FRET）,coomassie brilliant blue staining and western blotting showed that M?m and RBCm were fused successfully,with preserved the corresponding marker proteins of macrophage and erythrocyte,which could help HA-M@PB@（PC+ART）NPs to achieve immune escape and prolong the circulation time.Cell uptake assay showed that the biomimetic cell membrane coated nanoparticles has excellent immune escape function and inflammatory macrophage targeting ability.Results of hemolysis,coagulation and cytotoxicity assay showed that HA-M@PB@（PC+ART）NPs wouldn’t trigger hemolysis and coagulation,and the cytotoxicity was low,indicated that the constructed biomimetic nanosystems were safe and could be used in subsequent animal experiments.In vivo fluorescence imaging showed that HA-M@PB@（PC+ART）NPs were distributed in atherosclerotic sites in Apo E^-/-mice fed with high fat diet,achieving efficient targeting of the atherosclerotic lesion site.Blood routine test,blood biochemical analysis,and histopathology of maj or organs showed that HA-M@PB@（PC+ART）NPs did not cause side effect during the treatment,indicated HA-M@PB@（PC+ART）NPs have good biocompatibility,which leading to a good clinical transformation prospect.（3）Study the anti-atherosclerosis effect and mechanism of HA-M@PB@（PC+ART）NPs.To systematically investigate the anti-atherosclerosis effect and mechanism of HA-M@PB@（PC+ART）NPs,ROS scavenging assay,NO scavenging assay,lipid uptake assay,oil red O staining assay,western blotting and ELISA assay were used to evaluate the effect and mechanism of HA-M@PB@（PC+ART）NPs at the cellular level.Above results showed that HA-M@PB@（PC+ART）NPs had a good inhibitory effect on ROS and NO production and it can inhibit lipid influx by regulating the RONS/NF-κB/NLRP3/inflammation pathway.At the same time,HA-M@PB@（PC+ART）NPs can inhibit foam cell formation by reducing oxLDL uptake and deposition.Results of western blotting further indicated that HA-M@PB@（PC+ART）NPs could effectively promote cholesterol efflux by enhancing AMPK/mTOR/autophagy pathway.Apo E^-/-mice were fed with high-fat diet to establish atherosclerotic model.The therapeutic effect of HA-M@PB@（PC+ART）NPs was analyzed by oil red staining in gross and frozen sections of aorta,and result s showed that HA-M@PB@（PC+ART）NPs significantly inhibited the formation of atherosclerotic plaque in both 8 weeks of low-dosage treatment and 1 week of high-dosage treatment,indicating their excellent therapeutic effect.Plaque composition was analyzed by H&E staining,Masson staining and immunohistochemistry and results showed that treatment with HA-M@PB@（PC+ART）NPs significantly reduced the necrotic core of plaques,decreased the infiltration of inflammatory macrophages in plaques,and effectively incr eased the stability of plaques.ELISA and immunohistochemistry were used to investigate the in vivo therapeutic mechanism and results showed that HA-M@PB@（PC+ART）NPs alleviate atherosclerosis by reducing lipid influx and promoting cholesterol efflux.These results indicated that HA-M@PB@（PC+ART）NPs significantly alleviated atherosclerosis via simultaneously modulating lipid influx and cholesterol efflux and could be used for targeted therapy of atherosclerosis.In conclusion,we constructed a biomimetic n anosystem HA-M@PB@（PC+ART）NPs for targeted therapy of atherosclerosis based on the combinational effect ART and PC on lipid influx and cholesterol efflux of macrophages.It provides a safe and effective strategy for the application and functional design of natural compounds in atherosclerosis.