In Vitro And In Vivo Studies On Anti-atherosclerotic Activity Of An Orally Delivered Ros-responsive Multifunctional Nanotherapy

Atherosclerosis?AS?is a major cause of coronary heart disease,stroke,and peripheral vascular disease.As yet,the curative effect of AS drugs used in clinical is limited,always alone with some side effects.Oxidative stress is a common causative factor for atherosclerosis.Whether probucol-like antioxidants or traditional dietary supplementation did not have a positive effect on the treatment of atherosclerosis.Therefore,based on oxidative stress,exploring new anti-oxidation strategies has become the key role to prevent AS.Recently,our group demonstrated that a reactive oxygen species?ROS?responsive and eliminating nanoparticle?TPCD NP?had an excellent performance in the treatment of acute inflammatory diseases such as paw edema,peritonitis and chronic inflammatory diseases such as neutrophil asthma and AS by intravenous injection.Herein,on the one hand,we intend to study whether it has an anti-AS effect and potential regulatory mechanism by orally administered TPCD NP;on the other hand,we proposed to use TPCD as a ROS-responsive functional nanocarrier loaded with anti-inflammatory drug Darapladib?DA?to prepare a ROS-responsive multifunctional nanotherapy?TPCD-DA NP?and evaluate its treatment effect on AS and safety by oral administration.Methods1.The Synthesis and characterization of TPCDPrimarily,4-Hydroxy-Tempol?Tpl?and 4-?Hydroxymethyl?phenylboronic acid pinacol ester?PBAP?were both covalent link on?-cyclodextrin??-CD?.ROS-responsive material TPCD was synthesised.The structure and molecular weight of TPCD were confirmed by fourier-transform infrared spectroscopy?FT-IR?,nuclear magnetic resonance spectroscopy?¹H NMR?and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry?MALDI-TOF?.2.Preparation and characterization of TPCD NP and TPCD-DA NPThe DSPE-PEG2000 and lecithin both disperse in the aqueous phase,TPCD dissolved in methanol as the organic phase.The organic phase was dropwised into the aqueous phase with vigorous stirring at room temperature to obtain TPCD NP.In addition,TPCD-DA NP can be obtained by dissolving Darapladib and TPCD in methanol as the organic phase.The size,Zeta potential and PDI of the nanoparticles were measured by Malvern laser particle size analyzer?DLS?.Transmission electron microscope?TEM?were used to observe the morphology of the nanoparticles.3.ROS-responsive hydrolysis of TPCD NP and TPCD-DA NPThe hydrolysis effect of TPCD NP and TPCD-DA NP was detected by incubated with1 mM H₂O₂ or PBS at predetermined time point.The drug loading efficiency,entrapment efficiency and cumulative release curve of TPCD-DA NP were determined by high performance liquid chromatography.4.TPCD NP inhibits ROS production and ROS-induced cell apoptosis in vitroTPCD NP was firstly preincubated with RAW264.7,and then treated with PMA for 4 h.DCFH-DA ROS probe was used to explore the effect of TPCD NP on the inhibtion of intracellular reactive oxygen species.In an other way separated experiment,RAW264.7was treated with 200?M H₂O₂ for 8 h to evaluate the inhibitory effect of TPCD NP on apoptosis induced by high concentration of hydrogen peroxide.5.TPCD NP and TPCD-DA NP postpone ROS-induced cell senescence and inhibit cytokine secretionThe?-galactosidase staining kit was used to evaluate the effect of nanoparticles on hydrogen peroxide-induced cellular senescence on RAW264.7 and Caco-2 cells.In addition,the inhibitory effects of nanoparticles on MCP-1,TNF-?,and IL-1?were measured by ELISA.6.Anti-atherosclerosis evaluation of TPCD NP in vivoApo E^-/-mice at the age of 6-8 weeks were randomly divided into model group?high-fat diet?and TPCD NP group?25 mg/kg TPCD NP?respectively.After high-fat diets containing 20%fat,0.2%cholesterol,and 10%protein were fed one month,TPCD NP was administered by orally,once every 4 days and continuously for 11 weeks.The whole aorta was collected and oil red o staining was used to evaluate its effect on AS plaque area.7.Potential anti-atherosclerosis mechanism of TPCD NP in vivoApo E^-/-mice at the age of 6-8 weeks were randomly divided into 2 groups.One group was the ordinary feed diet group and the high-fat diet group;the other group was the high-fat diet group and TPCD NP group?25 mg/kg TPCD NP?by orally administration with once every 4 days and continuously for 11 weeks.Fecal samples were collected from all mice at the end of administration and the microbial population were detected by 16S r RNA gene sequencing technology.8.Anti-atherosclerosis evaluation of TPCD-DA NP in vivoApoE^-/-mice at the age of 6-8 weeks were randomly divided into 4 groups:model group?Saline?,free drug group?20 mg/kg Darapladib?,blank nanoparticle group?62.5mg/kg TPCD NP?and drug-loaded nanoparticles group?10 mg/kg TPCD-DA NP,corresponding to a TPCD NP dose of 62.5 mg/kg?respectively.After high-fat diets were fed2 months,different therapeutic agents was administered by orally,once every 4 days and continuously for 7 weeks.Samples were collected at the end of administration.Aortic oil red o staining was used to evaluate the effect of TPCD-DA NP on the AS plaque area and aortic root immunohistochemical staining was used to investigate the stability of plaque and the content of macrophages in plaque.The levels of LP-PLA2 in serum and inflammatory factors in different intestinal segments?middle intestines,lower intestines,and colon?homogenates were measured.9.Preliminary safety evaluation of TPCD NP and TPCD-DA NP in vivoBlood samples and main organs were collected at the end of administration.The effects of TPCD-DA NP on erythrocytes,white blood cells,platelets,hemoglobin,liver function,kidney function,and lipids profile were assessed and the main organs were stained with Hematoxylin-eosin?HE?to observe whether TPCD-DA NP had a side effect on them.Results1.Tpl and PBAP molecules were successfully conjugated on?-CD and a ROS-responsive material TPCD was successfully synthesized.TPCD NP and TPCD-DA NP were successfully prepared by modified nanoprecipitation method.The ROS-responsive hydrolysis of TPCD NP and TPCD-DA NP was more obviously in 1 m M H₂O₂ rather than in PBS.2.The drug loading of TPCD-DA NP was 16%and the encapsulation efficiency was85%detected by HPLC.The cumulative drug release rate at 5 h and 12 h was 82%and 96%respectively in 1 mM H₂O₂,indicated that drug was almost completely released.3.The results of in vitro cell experiments show that TPCD NP can significantly inhibit the production of intracellular ROS in RAW264.7 with dose-dependent manner.TPCD NP can also significantly inhibit high concentration of hydrogen peroxide-induced apoptosis.4.TPCD NP and TPCD-DA NP can postpone the senescence of RAW264.7 cells and Caco-2 cells induced by hydrogen peroxide.The content of MCP-1,TNF-?,and IL-1?in the cell medium supernatant was detected by ELISA.The results showed that TPCD NP and TPCD-DA NP can significantly reduce the secretion of inflammatory factors in cells and TPCD-DA NP group had a better efficiency.5.The results of in vivo efficacy evaluation experiments show that TPCD NP and TPCD-DA NP therapy can significantly reduce the area of aortic plaque in ApoE^-/-mice and TPCD-DA NP group had a better efficiency.In addition,the content of macrophages in the aortic root of mice was lower and the plaque stability was better in the TPCD-DA NP treatment group.6.Apo E^-/-mice gut microbiota test show that high-fat diet will change the composition of gut microbiota and reduce the abundance of gut microbiota.TPCD NP intervention can reverse the decreased gut microbiota abundance caused by high-fat diet.7.The results of preliminary safety evaluation experiments in vivo showed that after oral administration of TPCD NP or TPCD-DA NP,the body weight,blood routine,liver function,kidney function,and blood lipid profile of the mice were not significantly different from those of saline group.The main organs of HE staining results showed that the nanoparticles did not cause pathological changes in the organs after intervention.Conclusions1.TPCD NP were successfully constructed by the modified nanoprecipitation method based on ROS-responsive material TPCD.Similarly,TPCD-DA NP was prepared loading with anti-inflammatory drug Darapladib.2.In vitro cell experiments prove that TPCD NP or TPCD-DA NP have anti-inflammatory and anti-apoptotic effects.Additionally,they can also inhibit the increase of intracellular reactive oxygen species and cell senescence induced by oxidative stress.3.In vivo anti-atherosclerosis experiment demonstrated that orally delivered TPCD NP or TPCD-DA NP can significantly inhibit the aortic plaque area and the TPCD-DA NP therapy has a better treatment effect.In addition,TPCD NP can improve the reduction of gut microbiota abundance induced by high-fat diet to meet the effect of reducing the area of aortic plaque and realize the regulation of AS development process.4.The preliminary safety evaluation studies in vivo demonstrated that TPCD-DA NP can be used as a relatively safe nanotherapy drug for oral administration.In summary,we have prepared a new oral therapy TPCD-DA NP by combining the anti-inflammatory drug Darapladib and the ROS-responsive material TPCD,which is expected to be used as a safe and synergistic nanotherapy for the treatment of atherosclerosis related diseases.On the one hand,we proved that TPCD NP has a good prevention and treatment effect on AS and it may be to regulate AS by improving the gut microbiota abundance by orally delivered.