Targeted Therapy Of Liposome Nano-drug Delivery System In Homocysteine-induced Atherosclerosis

Cardiovascular disease（CVD）cause by atherosclerosis is a main threaten for human health.Hyperhomocysteinemia（HHcy）is an independent risk factor for atherosclerosis,with close link between homocysteine（Hcy）and DNA methylation modifications,DNA methylation playing an important role in the pathogenesis of atherosclerosis.In this study,we designed a macro-liposome nanoparticle by hybridizing liposome with macrophages membranes（M?m）to encapsulate hydroxysafflower yellow A（HSYA）.Assertion of Hyaluronic acid on the macro-liposome was adopted to endow nanodrug targeting ability.In vitro results showed that the prepared macro-liposome complexes significantly inhibit Atg13 DNA methylation,while enhanced autophagy of macrophages to promote cholesterol efflux.In vivo studies have shown that the HA modification and the macro-liposome make Lipo NPs an ideal decoy for targeting plaques.In addition,the hybridization of macro-liposomes can prolong blood circulation time,and the combination of HA can improve its targeting ability,to achieve optimal HSYA accumulation at the region of atherosclerotic plaques.In summary,the nano-drug delivery system with high immune escape capability can plaque-targeting and accumulate in the plaque region,thereby providing a feasible therapeutic strategy for efficient atherosclerosis therapy.The research of this paper mainly includes the following three aspects:1.Construction of cell level model and screening of HSYA drug concentrationObjective:Hcy successfully constructed a macrophage autophagy level reduction model at the cellular level and screened the optimal concentration of HSYA on macrophages.Methods:Mouse macrophages（RAW264.7）were cultured in vitro,Meth Primer bioinformatics analysis（http://www.urogene.org/methprimer/）,nested landing methylation specific PCR（nMS-PCR）,Western blot was used to detect the expression of LC3-II protein under different intervention conditions,fluorescence imaging of oxidized low-density lipoprotein was used to detect the phagocytosis of oxidized low-density lipoprotein by cells after different treatment,Oil Red O staining was used to detect the internalization of oxidized low-density lipoprotein by macrophages after Hcy intervention,and MTT assay was used to detect drug toxicity.Results:After Hcy intervention,the Cp G island of Atg13 was detected by Meth Primer bioinformatics analysis.After Hcy intervention,the methylation level in macrophages increased,while the protein level of LC3-II decreased.Fluorescence imaging showed that red fluorescence was significantly enhanced,and the internalization of oxidized low-density lipoprotein increased.MTT was used to screen the highest safe concentration of HSYA.nMS-PCR and Western blot showed that 50μM HSYA could reduce the methylation level of Atg13 DNA and increase the expression of LC3-II protein.Fluorescence imaging and Oil Red O staining showed that 50μM HSYA could reduce the internalization of oxidized low-density lipoprotein.Conclusion:100μM Hcy treatment of macrophages for 24 h can increase Atg13 DNA methylation level and reduce LC3-II protein expression;the optimal concentration of HSYA on macrophages was 50μM.2.Characterization and properties of biomimetic nanomedicinesObjective:To prepare HSYA-encapsulated macrophage membrane and artificial liposomes,and to detect their physical properties.Methods:Liposomes were prepared by film dispersion method,and hybrid liposomes were prepared by stirring method.The size and morphology of liposomes and hybrid liposomes were observed by transmission electron microscopy（TEM）.Confocal laser scanning microscopy（CLSM）,Coomassie brilliant blue and Western blot were used to detect the hybridization of liposomes and macrophage membranes.The encapsulation efficiency of Lipo NPs encapsulated HSYA and the release rate of HSYA were detected by UV-visible spectrophotometry.The zeta（ζ）potential and dynamic light scattering（DLS）were used to detect the positive and negative potentials of NPs,the particle size,and the stability of HA-ML@HSYA NPs.CLSM was used to detect the adhesion of NPs to endothelium,and confocal microscopy was used to detect the immune escape ability,targeting and lysosomal co-localization of NPs.The half-life of NPs blood circulation and the targeting of plaques were detected by a small animal multimodal imaging system.The biocompatibility of NPs was detected by MTT assay,hemolysis assay and coagulation assay.Results:HSYA-encapsulated hybrid liposomes were successfully prepared by film dispersion method and stirring method.TEM images showed that the uniform distribution of lipid NPs was about 100 nm in diameter.CLSM,Coomassie brilliant blue and Western blot showed that the macrophage membrane was successfully hybridized on the artificial liposomes.Zeta（ζ）potential and dynamic light scattering（DLS）detection showed that theζ-potentials of ML@HSYA NPs and HA-ML@HSYA NPs were-41.50 m V and-34.36 m V,respectively,and the diameter was about 122 nm and 142 nm.HA-ML@HSYA NPs had good stability in 10%DMEM.However,we found that the particle size of HA-ML@HSYA NPs increased with time in 10%PBS,and we speculated that the increase in the particle size of HA-ML@HSYA NPs was due to the formation of protein crowns.In addition,in vitro simulated release experiments showed that ML@HSYA NPs were p H dependent.CLSM detection found that the macrophage membrane gave the bionic nano-drug good immune escape ability,and hyaluronic acid（HA）gave the nano-drug good targeting.They worked together to escape the degradation of lysosomes.The detection of small animal multimodal imaging system showed that macrophage membrane and hyaluronic acid could effectively prolong the circulation half-life of NPs in blood and increase their targeting in blood.It was found that HA-ML@HSYA NPs had good biocompatibility by MTT assay,hemolysis assay and coagulation assay.Conclusion:HSYA coated liposome spheres with hybrid macrophage cell membrane were prepared successfully.The particle size was about 100 nm,and the potential was about-34.36m V,which showed good biocompatibility.3.The pharmacodynamic evaluation and mechanism of HA-ML@HSYA inhibiting ox LDL uptake in vitroObjective:To investigate the efficacy and mechanism of HA-ML@HSYA in inhibiting ox LDL uptake in vitro.Methods:The methylation level of Atg13 was detected by nMS-PCR.The expression of ATG13,LC3-II and p62 protein was detected by Western blot.The expression of LC3-II and p62 in macrophages after intervention with different drugs was detected by CLSM（green fluorescence labeling）.The formation of autophagosomes in macrophages after intervention with different drugs was observed by transmission electron microscopy（TEM）.The CLSM experiment of fluorescently labeled oxidized low-density lipoprotein was used to detect the phagocytosis of oxidized low-density lipoprotein by mouse macrophage cells after different treatments.Oil Red O staining was used to detect the internalization of oxidized low-density lipoprotein by macrophages after intervention with different drugs.RNA-sequencing（RNA-seq）was used to analyze the mechanism of HA-ML@HSYA NPs in macrophages.Results:nMS-PCR showed that HA-ML@HSYA NPs could effectively reduce the methylation level of Atg13,and Western blot showed that HA-ML@HSYA NPs could effectively up-regulate LC3-II and down-regulate p62 protein level.The results of CLSM were consistent with those of Western blot.TEM detection showed that autophagosomes in the Hcy group were significantly reduced compared with the control group,and HA-ML@HSYA NPs were significantly increased after treatment.Oil Red O staining showed that the internalized ox LDL in the Hcy group was significantly increased compared with the control group,and HA-ML@HSYA NPs were significantly reduced after treatment.RNA sequencing analysis showed that the molecular functions of hemi methylated DNA and histone demethylase were abundant.There were 166 differential genes between HMD+Saline and HMD+HA-ML@HSYA NPs groups,and 61 differential genes between Saline group and HMD+Saline group.There were 20identical differentially expressed genes（DEGs）between the two comparisons（HMD+Saline and HMD+HA-ML@HSYA NPs and Saline group and HMD+Saline group）.Conclusion:HA-ML@HSYA NPs can effectively promote the autophagy of macrophages and inhibit the uptake of ox LDL by macrophages by inhibiting the DNA methylation level of Atg13 in macrophages.4.The anti-atherosclerotic efficacy evaluation and mechanism study of HA-ML@HSYA NPs in vivoObjective:To investigate the efficacy and mechanism of HA-ML@HSYA NPs against atherosclerosis in vivo.Methods:Six-week-old male Apo E^-/-mice were fed with HMD for 2 months.After that,normal Saline,rapamycin（RAP）,HSYA and HA-ML@HSYA NPs were intravenously injected twice a week for 12 weeks.After treatment,Apo E^-/-mice were sacrificed and blood,aorta,and major organs（heart,liver,spleen,lung,and kidney）were taken for subsequent experiments.Aorta,aortic root,aortic arch,and abdominal aorta were stained with Oil Red O to detect plaque area.Hematoxylin-eosin（H&E）staining was used to detect plaque necrosis area and tissue structure of non-targeted organs.Masson staining was used to detect the proportion of collagen fibers in plaques.Immunohistochemical experiments were used to detect the stability and internal mechanism of plaques（F4/80,MMP-9,CD31⁺,α-SMA,ATG13,LC3-II,p62）.Non-targeted proteomics was used to analyze the changes of metabolites between high methionine diet and treatment group,and intestinal microbiome was used to detect the changes of intestinal flora between high methionine diet and treatment group.Results:ORO staining clearly showed that HA-ML@HSYA NPs significantly reduced the plaque area of aortic root,aortic arch,and abdominal aorta.The necrotic core area of mice treated with RAP and HA-ML@HSYA NPs was significantly reduced.HA-ML@HSYA NPs treatment effectively reduced the number of macrophages infiltrated in the plaque（about 10%compared with the model group）and the expression level of MMP-9.Masson trichrome staining showed that the collagen content around the plaque in the HA-ML@HSYA NPs treatment group was significantly increased,resulting in an increase in the thickness of the fibrous cap.Immunohistochemical experiments showed that after HA-ML@HSYA NPs treatment,CD31⁺neovascularization in the atherosclerotic plaque area was significantly reduced.In addition,α-SMA staining showed that HA-ML@HSYA NPs could increase the number of VSMCs.Immunochemical analysis showed that HA-ML@HSYA NPs promoted the expression of autophagy genes Atg13 and LC3-II and inhibited the expression of p62.Functional enrichment analysis of non-targeted metabolomics showed that ABC transporters were significantly present in KEGG,and the high level of 2,3-Dinor-Txb2,a metabolite that promotes the development of atherosclerosis,was reversed in the model group by HA-ML@HSYA NPs treatment.Conclusion:HA-ML@HSYA NPs can effectively promote macrophage autophagy by inhibiting the methylation level of Atg13 and inhibit the development of atherosclerosis in mice.