Construction Of Apigenin-loaded Hydrogen Dioxide Sensitive Nanoparticles And Evaluation Of Their Preliminary Anti-tumor Effect

With the development of high-throughput screening technology for drugs,a large number of active compounds for tumor therapy have been screened out,but most of the active compounds selected have high molecular weight and high hydrophobicity,which makes it unable to enter clinical applications.As an active substance,apigenin(API)has various pharmacological activities,such as anti-tumor,anti-inflammatory,anti-oxidation,etc.It has good market application prospects,but there are also problems of poor drug-forming properties due to poor water solubility.A large number of studies have confirmed that the drug nanotechnology can improve the poor dispersibility of the insoluble active substances,and has become a hot spot in pharmaceutics research.This project constructed a novel nano-drug system to solve the problem of poor solubility and low dissolution of the insoluble apigenin.The main research contents are as follows:1.Synthesis of Oxidized dextran(Oxi-Dex),a novel nano-carrier material with H2O2 sensitivity.The synthesis of Oxi-Dex was divided into two steps.Firstly,pinacol boronate ester(PBE)and N,N’-carbonyldiimidazole(CDI)react to produce imidazoyl carbamate(IC);secondly,the dextran and IC were synthesized to Oxi-Dex under the catalysis of dimethylpyridine(DMAP),And the Oxi-Dex was characterized by IR and NMR.On this basis,the factors affecting the synthesis of Oxi-Dex were optimized by single factor experiments.The optimization results showed that the number and effect of water washing were the key factors affecting the first step reaction.Washing more than 6 times,until the organic phase solution becomes colorless and transparent.Secondly,the molar ratio of PBE to CDI was also the main factor affecting the synthesis of IC.The experimental results showed that when the molar ratio of PBE to CDI was 1:2,the yield of IC was the highest,reaching 97.98%.The molar ratio of dextran to IC and reaction time were the main factors affecting the second step.The results showed that when the molar ratio of dextran to IC was 1:128 and reaction time was 24 h,the amount of Oxi-Dex was the highest.2.Synthesis of Oxi-Dex nanoparticles and optimization of synthesis process.In this study,The Oxi-Dex was used to prepare Oxi-Dex nanoparticles by ultrasonic combined with solvent evaporation method.On this basis,the preparation process of nanoparticles was systematically optimized.The results showed that the optimum conditions for the synthesis of nanoparticles are as follows:the volume ratio of oil phase to water phase was 1:3,the volume ratio of oil phase to water phase was1:13.3,the ultrasonic power was 50%,and the ultrasonic time was 180 s.Under the optimum conditions,the Oxi-Dex nanoparticles were 156.7±0.02 nm,the PDI was 0.034±0.01,and the Zata potential was-8.7±0.1 mV.In addition,the results of stability experiments showed that the stability of nanoparticles was good when the nanoparticles were placed at 4 0C for 30 days.In addition,the vitro toxicity test of nanoparticles showed that the survival rate of tumor cells was higher than 80%in the range of 0-300 ug·mL-1,which proved that Oxi-Dex was less toxic and had good safety when used as nanocarriers.3.Preparation of apigenin-loaded oxidized dextran nanoparticles.On the basis of preparation technology of blank Oxi-Dex nanoparticles,the preparation process of apigenin-loaded Oxi-Dex nanoparticles was optimized with drug loading and particle size as evaluation indexes.The results showed that the optimum parameters for preparation of Apigenin-loaded Oxi-Dex nanoparticles were as follows:Apigenin dosage was 1 mg,the ratio of oil phase to water phase was 1:3,the volume ratio of oil phase to water phase was 1:13.3,the ultrasonic power was 50%,and the ultrasonic time was 180 s.Under the optimized conditions,the particle size of the apigenin-loaded Oxi-Dex nanoparticles was 178.7±0.01 nm,the PDI was 0.056±0.03,the Zata potential was-8.9±0.1 mV,the drug loading rate of apigenin was 3.69%(36.85 ug.mg-1),and the encapsulation rate was 55.27%.Moreover,the prepared Apigenin-loaded Oxi-Dex nanoparticles have good sensitivity to H2O2.4.To evaluate the killing effect of apigenin-loaded Oxi-Dex nanoparticles on cancer cells in vitro.Apigenin-loaded Oxi-Dex nanoparticles have a good killing effect on Hela cells.The IC50 value of Apigenin-loaded Oxi-Dex nanoparticles was 42.6 μg·mL-1.The killing effect of Apigenin on Hela cells was lower than that of the above nano-loaded system.The IC50 value of Oxi-Dex nanoparticles was 50.3 ug mL-1.The results showed that the inhibitory effect of Apigenin-loaded Oxi-Dex nanoparticles on Hela cells was better than that of Apigenin,especially with the increase of drug concentration.Cell apoptosis experiments showed that compared with apigenin,apigenin-loaded Oxi-Dex nanoparticles could induce higher levels of apoptosis.In conclusion,in this study,Oxi-Dex was used as nano-carrier material and apigenin was used as active substance,apigenin-loaded Oxi-Dex nanoparticles were prepared by ultrasonic combined with solvent evaporation method.The nano-drug delivery system was sensitive to H2O2 and can passively target tumor microenvironment.It is expected to achieve targeted and efficient treatment of cancer.