Preparation Of Hydrophilic HA-ABA Nano Drug Delivery System And Its Anti-Tumor Effect In Vitro

BackgroundAbscisic acid(ABA)is a plant hormone with a sesquiterpene structure and is widely distributed in various higher plants.It not only plays an important role in the life cycle of plants,but also have a significant regulatory effect on immune cells,cardiovascular tissues and cells,stem cells and pancreatic cells.Etc.Recent studies have found that ABA can inhibit the proliferation and induce apoptosis of a variety of cancer cells,and is expected to become a new generation of antitumor drugs.Esophageal cancer is a malignant tumor of the digestive tract that is not easy to detect at an early stage.About 200,000 people worldwide die of esophageal cancer every year.In China,the incidence of esophageal cancer is among the top5 in the world,posing a great threat to people’s lives and health.Surgery,chemotherapy,and radiotherapy are the main treatment methods for esophageal cancer.The use of traditional Chinese medicine has shown some certain clinical advantages,mainly in the aspects of improving postoperative immunity,reducing side effects,and suppressing tumor recurrence and metastasis.The anti-esophageal cancer effect of ABA has not been reported so far.Therefore,ABA was used to prepare a hydrophilic nanoparticle and explore its effect on esophageal cancer cells in order to provide new ideas for the treatment of esophageal cancer.ObjectiveAbscisic acid,as an active ingredient of traditional Chinese medicine,also has a certain inhibitory effect on the value-added of esophageal cancer cells.In order to improve the effect of ABA,this article creatively modified the abscisic acid structure by chemical method with water-soluble drug carrier hyaluronic acid(HA).The preparation of a new drug delivery system HA-ABA can achieve the purpose of increaseing drug solubility and increasing drug targeting,and greatly improve the bioavailability of abscisic acid,thereby improving the efficacy of ABA.Methods(1)Preparation of HA-ABA nanoparticles:Intermediate HA-ADH is synthesized from HA and Adipic dihydrazide(ADH).and then reacted with ABA to prepare the final product HA-ABA.HA-ABA nanoparticles were further prepared by phase inversion and ultrasound.1H-NMR,FT-IR.Size distribution and Zeta potential and TEM were used to investigate and verify HA-ABA.Establish the content determination method of HA-ABA,and use this to detect its content and stability.(2)Antitumor effects in vitro:The experiment was divided into four groups:Control group.HA group.ABA group and HA-ABA group.After human esophageal cancer cells KYSE-30/70 were treated with medicine,the cell viability was detected by MTT and CCK-8;The proliferation ability of cells was tested by EdU;The migration and invasion of esophageal cancer cells were verified by cell scratches.Transwell and Invasion experiments;Plate cloning experiment was used to verify the ability of cell clone formation;The apoptosis ability of cells was detected by TUNEL;Flow cytometry was used to examine the effects of HA-ABA on the apoptosis and cycle of esophageal cancer cells,and the relationship between apoptosis and autophagy was explored;The expression of apoptosis,autophagy,and cycle-related proteins were measured by Western blot.Results(1)Characterization results of HA-ABA nanoparticles:1H-NMR spectrum shows that the loading degree of HA-ABA is about 21.4%;FT-IR spectra verify the chemical structure of HA-ABA;HA-ABA nanoparticles have an average particle diameter of 168.3nm.a PDI of 0.278 and a potential of-26.9mV,and show smooth and no adhesion;The content of HA-ABA nanoparticles is 19.7%.and the release rate of HA-ABA in vitro is the highest at 24h.(2)Results of in vitro antitumor effects:The results of MTT and CCK-8 experiments showed that the survival rate of human esophageal cancer cells KYSE-30/70 in the HA-ABA group was significantly lower than that of the control group;EdU test showed that the proliferation number of cells which were treated with HA-ABA was greatly different from that of each control group.It can be seen from cell scratches and Transwell experiments that the cell migration rate and migration number of each administration group were obviously lower compared with control group,and HA-ABA significantly inhibited tumor cell migration ability;Invasion experiments indicated that the invasion of esophageal cancer cells was also inhibited by HA-ABA;In plate cloning experiment,contrasted with control group,the number of cell clones formed in administration group was reduced,and the cloning ability was weakened;The TUNEL experiment is consistent with the flow cytometry detection of apoptosis,suggesting that HA-ABA has a better pro-apoptotic effect,and inhibition of protective autophagy can markedly increase its apoptosis-inducing effect;Cell cycle tests proved that HA-ABA can block cells in the G2/M phase;Western blot manifested that HA-ABA can reduce the expression level of autophagy protein P62,and up-regulate the expression level of Beclinl and LC3B;At the same time,the apoptosis proteins Bax/Bcl-2 and Bad/Bcl-xl are regulated,and the ratio is significantly increased.The expression of Cleaved Caspase-3,Cleaved Caspase-9,and Cleaved PARP is obviously increased;HA-ABA can down-regulate the expression of cyclins CyclinB1 and CDK1.Conclusion(1)HA-ABA nanoparticles have good hydrophilicity and high stability,and are a new dosage form antitumor drug with broad medical prospects.(2)HA-ABA nanoparticles can significantly inhibit the survival,proliferation,migration and invasion of human esophageal cancer cells KYSE-30/70.(3)HA-ABA nanoparticles can induce human esophageal cancer cell KYSE-30/70 to undergo apoptosis,autophagy,and cycle arrest.