Rosuvastatin-loaded Biomimetic Nano Prussian Blue Targeted Therapy For HHcy Induced Atherosclerosis And Its Mechanism

Rosuvastatin（RVS）with good anti-inflammatory and lipid-lowering functions,is the first-class clinic drug for the treatment of atherosclerosis.However,RVS for hyperhomocysteinemia caused atherosclerosis therapy faces the challenges of high oral dose,poor target,and toxic side effects for long term usage.In this study,we applied nanotechnology to load RVS on the Prussian blue nanoparticles（PB NPs）and camouflaged them with macrophages membrane（MPR NPs）to improve bioavailability and targeting capacity to plaque lesions for the treatment of HHcy induced atherosclerosis.In vitro experiments showed that the prepared MPR NPs scavenged ROS and reduced lipid uptake,while attenuated inflammatory response by inhibiting homocysteine（Hcy）induced macrophage pyroptosis through regulating TLR4/NLRP3signaling pathway.Meanwhile,in vivo experiments indicated that MPR NPs with good biological safety effectively treated atherosclerosis in Apo E^-/-mice fed a high methionine diet,reduced blood Hcy levels,and remodeled the abundance of beneficial gut microbes.In summary,the targeted delivery system of RVS with the assistance of bionic PB NPs effectively treated Hcy-induced atherosclerosis,which provides a theoretical basis for the clinical translation of nanodrugs.The research in this paper mainly includes the following three aspects:1.Construction and characterization of biomimetic nanosystemObjective To construct the bionic nanosystem and characterize its construction success.Methods Transmission electron microscopy（TEM）was observed the morphology and particle size of PB NPs.Ultraviolet visible spectrophotometry was measured the loading efficiency,entrapment efficiency and cumulative release of PB NPs.The expression of M（?）m characteristic proteins on MPR NPs was detected by Coomassie brilliant blue staining and Western blot,and the co-localization of PB NPs and M（?）m were detected by confocal laser scanning microscopy（CLSM）.Zeta（ζ）potential and dynamic light scattering（DLS）were used to measure theζpotential and particle size.Fourier transform infrared spectroscopy（FT-IR）was used to detect the specific chemical bond common to PR NPs and RVS.The immune escape capacity of NPs,NPs recruitment capacity of endothelial cells,NPs uptake capacity of cells,and subcellular localization of NPs were detected by CLSM.The biocompatibility of NPs was determined by MTT and hemolysis/coagulation test.Results M（?）m successfully wrapped on the surface of PR NPs to form MPR NPs.TEM showed that MPR NPs were cubic with clear membrane structure around them,and MPR NPs retained M（?）m characteristic protein differentiation antigen cluster 11b（CD11b）.When the mass ratio of PB and RVS are 1:1,the loading efficiency（LE）and entrapment efficiency（EE）were 44.1%±0.1%and 88.2%±0.2%,respectively.CLSM showed that PB NPs and M（?）m were good co-location.DLS showed that the particle size of MPR NPs was 120 nm±14 nm,and theζpotential was about-14.58±0.28 m V.FT-IR results showed that PR NPs and RVS had the characteristic chemical bond of RVS,indicated that PB NPs successfully loaded RVS.CLSM showed that MPR NPs had the ability to escape the phagocytosis of immune cells and avoid lysosome clearance after entering macrophages.PB NPs encapsulated in M（?）m enhance the recruitment ability of endothelial cells.At the same time,MPR NPs had specific targeting ability to macrophages under the condition of co-culture of endothelial cells and macrophages.The results of in vitro release showed that MPR NPs promoted the release of RVS from MPR NPs under p H 6.8.In addition,MPR NPs had good biocompatibility.Conclusion Biomimetic nanodrug delivery system was successfully constructed.The system has the characteristics of appropriate particle size,p H response release,and excellent homologous targeting ability,immune escape ability and good biocompatibility.2.Anti-atherosclerosis effect and molecular mechanism of MPR NPs in vitroObjective To explore the mechanism of MPR NPs in vitro treatment of atherosclerosis caused by Hcy and evaluate the biocompatibility of MPR NPs.Methods Flow cytometry（FCM）was used to detect Reactive oxygen species（ROS）positive cell rate.CLSM and Oil red O（ORO）staining were used to determine the uptake and internalization of Oxidized-low density lipoprotein（ox-LDL）in macrophages.RNA sequencing（RNA-seq）detected differentially expressed genes（DEG）,KEGG signaling pathways,and biological processes of Gene ontology（GO）in macrophages after Hcy-induced.Western blot,immunofluorescence,si RNA transfection and ELISA were used to detect macrophage pyroptosis related proteins to explore the molecular mechanism of MPR NPs in the treatment of atherosclerosis.Results FCM showed that MPR NPs effectively reduced the rate of ROS positive cells,up-regulated ATP binding cassette subfamily A member 1（ABCA1）,ATP binding cassette subfamily G member 1（ABCG1）expression,and reduced ox-LDL uptake and internalization of macrophages.RNA-seq results showed that Hcy induced macrophages,up-regulated Toll like receptor 4（TLR4）,Nuclear transcription factor-κB（nuclear transcription factor-κB）,NF-κB),NOD like receptor thermal protein domain associated protein 3（NLRP3）signaling pathway and genes related to inflammatory response and oxidative stress.Gene set enrichment analysis（GSEA）map was also used to directly show the degree of differential expression.Western blot and immunofluorescence results showed that MPR NPs reversed the up-regulated protein expression TLR4,Myeloid differentiation primary response gene 88,（My D88）,NF-κB,NLRP3,Apoptosis-associated spotted proteins（ASC）,Pro-caspase1,Caspase1,C-GSDMD,Interleukin-1β（IL-1β）,and Interleukin-18（IL-18）induced by Hcy.Meanwhile,the levels of IL-1βand IL-18 in extracellular environment were also significantly reduced by MPR NPs.Conclusion MPR NPs can inhibit Hcy-induced pyroptosis of macrophages and reduce inflammation through the TLR4/NLRP3 signaling pathway.At the same time,MPR NPs can reduce ROS production in macrophages and inhibit lipid uptake and deposition.3.Efficacy,mechanism and safety evaluation of MPR NPs against atherosclerosis in vivoObjective To observe the anti-atherosclerosis effect of MPR NPs in vivo,and further explore its mechanism and safety.Methods 6-week-old male Apolipoprotein E(Apo E^-/-)knockout mice were fed High methionine diet（HMD）for 8 weeks and then randomly divided into 5 groups:The Control group,HMD group,PB NPs treatment group,RVS treatment group and MPR NPs treatment group.Each group were respectively injected by caudal vein twice a week for 10 weeks.After treatment,the mice were sacrificed.The blood,aorta,and major organs（heart,liver,spleen,lung,and kidney）were collected for follow-up experiments.Small animal multimodal imaging system to detect MPB NPs blood circulation half-life and plaque targeting.ORO staining was used to detect the percentage of plaque area in blood vessels.Hematoxylin-eosin（H&E）staining was used to detect the necrotic center core size in the plaques of Apo E^-/-mice in each group.Masson staining was used to detect the collagen fiber content in the plaques.Immunohistochemical staining of Mouse EGF-like module-containing mucin-like hormone receptor-like 1（F4/80）,Cluster of differentiation 31（CD31）,Matrix metalloproteinase-9（MMP-9）and alpha-smooth muscle actin（α-SMA）positive cell levels.The co-localization of NLRP3 and ASC in plaques and the expression of C-GSDMD in F4/80 labeled macrophages were detected by immunofluorescence.Serum levels of TC and TG were measured by Total cholesterol（TC）and Triglycerides（TG）kits.The biosafety of MPR NPs was evaluated by blood routine,biochemical analyzer,and organ H&E staining.Results MPB NPs extend the half-life of blood circulation and had the ability to target atherosclerotic plaque.HMD-fed Apo E^-/-mice treated with MPR NPs significantly inhibited aortic plaque formation and reduced the area of necrotic core within the plate.In addition,MPR NPs effectively reduced the number of F4/80⁺macrophages,the number of CD31⁺neovascular,the number ofα-SMA⁺smooth muscle cells and the expression level of MMP-9,and increased the content of collagen fibers.In addition,MPR NPs treatment reduced the elevated TC and TG levels caused by HHcy.At the same time,compared with the abnormality of Aspartate aminotransferase（AST）and Alanine aminotransferase（ALT）after RVS treatment,no significant abnormalities were observed in AST and ALT after MPR NPs treatment.In addition,there were no significant differences in H&E staining and blood routine of other components except the pathological changes of lipid infiltration in the liver of mice in the HMD group.Conclusion MPR NPs significantly inhibited plaque formation and increased plaque stability,and improved intestinal flora disturbance caused by high methionine diet.In addition,MPR NPs has good biological safety.