Construction Of PH-sensitive Modified Chitosan Nanoparticle Drug Delivery System And Its Anti-inflammatory And Anti-oxidation Effects

Cardiovascular disease is one of the diseases with the highest morbidity and mortality worldwide.Although the country's investment in it has increased year by year,the mortality rate has not decreased,posing a serious threat to human health.Atherosclerosis is the main cause of cardiovascular disease.The pathological process of atherosclerosis is a chronic inflammatory process involving the gradual accumulation of cholesterol deposits and the deposition of plaques in the arterial wall,eventually leading to arterial lumen narrow or even blocked.Objective: In this study,chitosan was amphiphilic modified with octenylsuccinic anhydride(OSA)to synthesize novel octenylsuccinylation modified chitosan(OSA-CS)to construct pH-sensitive OSA-CS amphiphilic polymer nanocarriers.Nanoparticle drug delivery system for releasing a drug in response to an acidic microenvironment of an inflammatory lesion was prepared,and the anti-inflammatory and anti-oxidative activity of the nanoparticle drug delivery system was evaluated.Methods:(1)Octylsuccinic anhydride(OSA)was grafted with chitosan by direct acylation to synthesize octenylsuccinyl-chitosan(OSA-CS).The structure of OSA-CS was characterized by nuclear magnetic resonance spectroscopy and infrared spectroscopy.The degree of substitution and solubility of OSA-CS were determined by acid-base titration and the critical micelle concentration was determined by pyrene fluorescence probe method.(2)Preparation of nanoparticle drug delivery system by encapsulation of curcumin(CUR)and quercetin(QUE)by solvent injection.The encapsulation efficiency and drug loading content of the nanoparticle drug delivery system were determined by UV spectrophotometry.The particle size,polydispersity and Zeta potential were determined by laser particle size analyzer,and the drug input,different pH conditions and stability of the nanoparticle drug delivery system were investigated.The morphology of the nanoparticle drug delivery system was observed by transmission electron microscopy.And the drug release characteristics in vitro were examined by dialysis method.(3)Cytotoxicity and cell viability were measured by CCK-8 method,and hemolysis and adhesion experiments were used to evaluate the blood compatibility of OSA-CS.(4)The anti-oxidation activity of the nanoparticle drug delivery system was evaluated by DPPH method.The cell uptake of CUR-OSA-CS was observed by fluorescence microscopy.Lipopolysaccharide(LPS)induced macrophage was used as an inflammatory model to investigate the ability of nanoparticle drug delivery system to scavenge reactive oxygen species(ROS).Results:(1)The octenylsuccinic anhydride was grafted chitosan to synthesize OSA-CS,and the degree of substitution increased with the increase of the molar ratio of octenylsuccinic anhydride and chitosan,and the degree of substitution was up to 38.89%.The critical aggregation concentration(CAC)of OSA-CS was 0.027 mg/mL and the smaller CAC guarantees the dilution stability of the nanoparticle drug delivery system in the blood circulation.(2)The loading efficiency of CUR and QUE loaded in OSA-CS could reach 14.4% and 12.1%,the encapsulation efficiency could reach 84.3% and 90.0%,the particle size was 171.7 and 189.8 nm,the polydispersity coefficient(PDI)was 0.145 and 0.181,and Zeta potential was-1.57,-1.74 mV,and CUR and QUE were co-loaded at 1:1 to prepare CUR+QUE-OSA-CS with the particle size of 166.6 nm,PDI of 0.197,and Zeta potential of-1.69 mV,indicating that the nanoparticle drug delivery system has a small particle size and a uniform particle size distribution.The environmental pH value has a great influence on the particle size and Zeta potential of the nanoparticle drug delivery system.When the pH value gradually decreases to 6.0,the surface charge of the nanoparticles was reversed from a negative potential to a positive potential,and the PDI is significantly increased,multi-peak distribution appeared,and the swelling and cleavage of nanoparticles was observed under TEM,showing good pH sensitivity.There was no significant change in the particle size of the drug-loaded nanoparticles in storage conditions and in serum,showing good stability.The drug release of CUR-OSA-CS and QUE-OSA-CS was affected by the pH of the release medium and CUR-OSA-CS and QUE-OSA-CS have the highest cumulative release in the acidic release medium of pH 6.0 for targeted delivery purposes,and release the drug slowly within 48 hours,with good sustained release properties.(3)Cytotoxicity tests of OSA-CS,CUR-OSA-CS,QUE-OSA-CS showed no cytotoxicity.Compared with free drugs,In addition,OSA-CS has no hemolysis and platelet adhesion,indicating that OSA-CS has better blood compatibility than chitosan(P<0.001).(4)The half-inhibitory concentrations of CUR-OSA-CS,Free CUR,QUE-OSA-CS,Free QUE,CUR+QUE-OSA-CS,and Free CUR+QUE on DPPH free radicals are 3.97,4.98,1.29,1.89,1.95,1.49?g/mL,respectively,indicating that the nanoparticle drug delivery system has higher antioxidant capacity than the free drugs(P<0.05).In addition,the results of cell uptake experiments showed that the nanoparticle drug delivery system had higher cell uptake rate than the free drugs(P<0.05).Moreover,CUR-OSA-CS,QUE-OSA-CS and CUR+QUE-OSA-CS inhibited the production of intracellular reactive oxygen species and inflammatory cell proliferation induced by LPS more effectively than free drugs(P<0.01),especially the nanoparticles loaded with two traditional Chinese medicine components had a synergistic effect(P<0.01),and exerted better anti-inflammatory and antioxidant effects.Conclusion: In this study,a pH-sensitive new soluble OSA-CS nano drug carrier with two Chinese herbal components was prepared,which had higher encapsulation efficiency and loading efficiency for curcumin and quercetin,and small particle size and uniform particle size distribution.It can fully play the synergistic effect of the combination of the two drugsad and the pH-sensitive intelligent response targeted delivery can reduce the damage of the drug to normal tissue cells,and OSA-CS can slowly and smoothly release the drug in response to changes in environmental pH,that can extend the action time,improve the anti-inflammatory and anti-oxidation therapeutic effect,reduce the side effects of the drug in the body.The above findings provide important experimental data for the clinical application of pH-sensitive sustained-release targeting agents for inflammatory microenvironments.