Construction Of Reactive Oxygen Responsiveness Particulate Delivery Systems For ARA290 And Their Applications In Antiatherosclerosis

BackgroundCardiovascular disease（CVD）,regarded as the a kill for human health,has become a serious threat to human health,and atherosclerosis（AS）is the basic process.At present,AS are treated by statins in clinic mainly.Aspirin is also used as an anti-inflammatory and anti-platelet activation drug in primary and secondary prevention of AS.They have played a certain role in clinical treatment,However,the course of AS can not be completely inhibited,and sideeffects is constantly emerging due to long-term administration.Therefore,it is significant to search novel effective anti-AS drugs and establish a long-term drug delivery system with low toxicity and side effects for the prevention and treatment of AS even CVD.AS is a chronic inflammatory disease induced by multiple factors,and the inflammatory response induced by oxidative stress in intima is the key to the initiation.Detailedly,reactive oxygen species（ROS）and oxidized lipoproteins activate inflammation in endothelial cells by damaging endothelial cells and stimulating endothelial cells to secrete a large number of inflammatory and chemokines after oxidative stress.Oxidative stress also plays an important role in the development of AS,such as impairing e ndothelial cell function through nitric oxide,promoting endothelial secretion of inflammatory factors through ROS.So inhibition of oxidative stress in vascular intima is an effective strategy to prevent and treat AS.ARA290 is a tissue protective polypeptide with 11 amino acids derived from erythropoietin（EPO）,that has been proved to be a potential anti-AS drug owing to outstanding anti-inflammatory,antioxidant and tissue repair effects.ARA290 loaded ROS responsive nanoparticle delivery system with ox-bCD was synthesized by nanoprecipitation self-assembly method,and its anti-AS efficacy in vivo and in vitro was observed.Methods1.The treatment of AS with ARA290 in vitro1.1 Effects of ARA290 on the production of ROS in macrophages under inflammatory conditionsRAW264.7 was stimulated by PMA for 2 h,and ARA290 with varied dose was added for co-incubation.ROS staining was performed with DCFH-DA after 2 h.Flow cytometry was used to detect intracellular ROS levels and fluorescence imaging was obtained.1.2 Effect of ARA290 on the secretion of inflammatory factors by macrophages under inflammatory conditionsMice macrophages were stimulated with LPS/IFN-γand incubated with ARA290 at different doses for 12 or 24 h.The inflammatory cytokines MCP-1,TNF-αand IL-1βwere detected by ELISA kit.1.3 Effect of ARA290 on oxidative-induced apoptosis of macrophagesRAW264.7were incubated with different doses of ARA290 for 2 h.After 7 h of incubation with H₂O₂,the cells were digested and collected.The protective effect of ARA290 on macrophage apoptosis induced by H₂O₂was detected by fluorescence probe staining and flow cytometry.1.4 Effect of ARA290 on MCP-1-induced macrophage migrationThe migration ability of RAW264.7 induced by MCP-1 was measured by Transwell assay at different concentrations of ARA290.1.5Effect of ARA290 on foam cells formed by macrophagesRAW264.7 was incubated with LPS（500 ng/mL）for 4 h.Different doses of ARA290and 50μg/mL ox-LDL were added to incubate for 36 h.The sample was fixedby 4%polyformaldehyde and stained by oil red for photography.1.6 Safety evaluation of high dose ARA290 on mouse macrophagesAfter 24 hours of incubation with different doses of ARA290 and RAW264.7,CCK-8kit was used to detect cell viability.2.Evaluation of anti-AS ability of ARA290 in vivoNine-week-old ApoE^-/-mice were fed with high-fat diet for 4 weeks.They were divided into three groups:control group,high-dose ARA290 group and low-dose ARA290group.ARA290 were given via i.p.administration for 10 weeks every day.After the mice were executed,blood routine and biochemistry tests were taken and the aortic plaque area was measured after stained with oil red.3.Construction of ARA290 loaded nano-delivery system3.1 Synthesis and characterization of materialsAfter activation with N,N’-carbonyl diimidazole（CDI）and dissolution withβ-CD and4-dimethylaminopyridine（DMAP）in anhydrous DMSO,the hydroxymethyl benzoborate ester（PBAP）was precipitated in deionized water at 25°C for 24 h.After three times of water washing,the white powder product was obtained.The chemical structure of PBAP was determined by Fourier transform infrared spectroscopy（FTIR）and nuclear magnetic resonance（NMR）.3.2 Preparation of ARA290 loaded ROS responsive nanoparticleARA290 loaded nanoparticles were prepared by nano-precipitation method.Lecithin anhydrous ethanol solution and phospholipid ethanolamine-polyethylene glycol（DSPE-PEG2000）solution were mixed and stirred at 65°C for 1 h to act as water phase.ARA290 or FITC-ARA290 were dissolved in different organic solutions,and then mixed with methanol solution within ox-bCD as oil phase.Oil phase was dropped into water phase at 0°C and stirred for 2 hours,then ARA290/ox-bCD and FITC-ARA290/ox-bCD nanoparticles were obtained during centrifuging at high speed and freezing-drying after washing with deionized water for 3 times.The particle size was measured by Malvine dynamic light scattering after redispersed in deionized water.The drug loading and entrapment efficiency were measured by fluorescence spectrophotometry after dissolved by methanol.Nanoparticles suspensionin deionized water was dripped onto copper mesh and mica sheet for TEM and SEM photographs,respectively.3.3 Hydrolysis and release of ARA290 loaded nanoparticles in vitroAppropriate FITC-ARA290/ox-bCD nanoparticles was suspended in PBS solution with different concentration of H₂O₂ and placed in incubator at 37°C.The supernatants were achieved by centrifugation at regular intervals and equivalent fresh solution was supplemented.The supernatants were stored in dark place.The drug release was measured by fluorescence spectrophotometer and the cumulative release curve was drawn.4.Evaluation of anti-AS ability of ARA290 loaded nanoparticles in vivo4.1 Evaluation of anti-AS ability of ARA290 loaded nanoparticles in vivoNine-week-old ApoE^-/-mice were fed with high fat diet for 4 weeks.They were divided into 4 groups:model group,ARA290 group,blank nanoparticles group,ARA290loaded nanoparticles group.After 10 weeks of i.p.administration,the mice were executed.Blood routine and biochemistry tests were taken and the aortic plaque area was measured after stained with oil red.4.2 Preliminary biosafety evaluation of ARA290 loaded nanoparticlesEight-week-old balb/c mice were injected with different concentrations of ARA290loaded nanoparticles and 0.1 mL PBS sterile solution via tail vein.After one week of normal feeding,blood was collected to measure biochemistry,blood routine and organ index.Result1.ARA290could effectively inhibit the inflammatory and production of ROS in mouse macrophages.Flow cytometry and ELISA kit showed that ARA290 could effectively inhibit the secretion of inflammatory-related cytokines such as IL-6,MCP-1and TNF-αin RAW264.7,as well as the production of ROS in inflammatory macrophages.2.ARA290 could inhibit the apoptosis of macrophages in mice under inflammation was confirmed by flow cytometry.3.ARA290 could effectively inhibit macrophage migration and foam cell formation in mice.Cell staining showed that ARA290 inhibited the migration of macrophages induced by MCP-1,uptake of ox-LDL and the formation of macrophage derived foam cells.4.ARA290 could effectively inhibit aorta plaque area in mice.In the long-term administration of ApoE^-/-mice model,ARA290with low dose could effectively inhibit the growth of plaque without obvious side effects.5.ARA290 had highly safety in mouse macrophages.Even if the extremely high dose of ARA290（1000μg/mL）was incubated with RAW264.7 for 24 h,apoptosis could not induced significantly.6.Carrier materials were successfully synthesized and their structures were characterized.The ox-bCD modified by PBAP was synthesized fromβ-CD.It was found that five PBAP units were bound to oneβ-CD by IR and NMR detection.7.The ROS responsive nanodelivery system of ARA290 was successfully constructed and characterized.Ox-bCD is insoluble in water and soluble in methanol,yet ARA290 is soluble in water and acidic organic solvents.In order to mixARA290with the methanol solution of ox-bCD forformation of oil phase,the solvent was constantly changed.The ARA290/ox-bCD nanoparticles with high drug loading were synthesized by nanoprecipitation self-assembly method.8.ARA290 loaded ROS responsive nanoparticles could reduce aortic plaque areaCompared with ARA290 group and blank nanoparticles group,ARA290nanoparticlesgroup exhibited more remarkable effect of reducing the aortaplaque area in ApoE^-/-mice during long-term administration.Conclusion1.Both in vivo and in vitro experiments showed that ARA290 had anti-AS effect.2.The AR290 loaded nano-delivery system based on ox-bCD was successfully constructed.3.In vivo experiments showed that ARA290 loaded ROS responsive nanoparticles had improved the anti-AS effect of ARA290effectively.