Construction Of Redox-sensitive Liposomes Modified By Glycyrrhetinic Acid And Evaluation Of Antihepatocellular Carcinoma Activity

Liver cancer is a common clinical malignant tumor,which seriously threatens people’s life and health.Especially in China,the mortality rate of liver cancer has always been high.The reason for the high mortality rate of liver cancer is that its disease is insidious and difficult to be detected in early stage.When patients show clinical symptoms,they are often in the middle and late stages,and miss the good time for treatment.Furthermore,there is a problem of difficulty in using drugs in the clinical treatment of liver cancer.The main reason is that it is difficult for drugs to effectively work on liver cancer tissues and have toxic side effects on normal human tissues that cannot be ignored.Therefore,it is urgent to build a drug delivery system to effectively deliver drugs to hepatocellular carcinoma tissues.Liposomes are excellent drug carriers and are favored by researchers because of their biocompatibility and ease of functionalization.If functional modifications are made to confer liposome targeting and tumor microenvironment responsiveness,precise delivery and effective release of anticancer drugs can be achieved.This can improve drug efficacy and reduce toxic side effects.Glycyrrhetinic acid（GA）is an active ingredient in the traditional Chinese medicine licorice.Studies have shown that this component can be effectively recognized by GA receptors overexpressed on the surface of hepatocellular carcinoma cells,and modifying liposomes with GA means conferring active targeting effects on liposomes liver.In addition,due to the special reducing environment in tumor cells,disulfide bonds can be broken.Therefore,modifying liposomes with molecules containing disulfide bonds can cause the liposomes to break the bonds in tumor cells,resulting in structural damage and release of drugs,which can effectively concentrate the drugs in tumor cells.Therefore,in this study,we constructed a drug-loaded liposome delivery system through targeted reductive responsive functional modifications to investigate its usefulness for the treatment of hepatocellular carcinoma.Objective:A bifunctional ligand with active liver targeting and intracellular tumor reduction response was constructed to modify the drug-loaded liposomes.The drug was precisely delivered to the liver focal tissues and rapidly responded to the hepatocellular carcinoma cells to release the drug in large quantities.It effectively increases the drug concentration in liver cancer cells while reducing the amount of drug in other normal tissues.The ultimate goal is to improve the bioavailability of drugs and reduce the toxic side effects on normal tissues of the body,so as to improve the efficacy of drugs against liver cancer.Methods:Firstly,GA-SS-NH₂was synthesized by linking glycyrrhetinic acid（GA）molecule with liver active targeting and cystamine through amide reaction.Then Suc-Chol was synthesized by modifying membrane cholesterol to introduce carboxyl group.Finally GA-SS-NH₂was linked with Suc-Chol through amide reaction again to synthesize GA-SS-Chol.The bifunctional ligand GA-SS-Chol molecule with hepatic active targeting and tumor intracellular responsiveness was synthesized.The intermediates and products were structurally verified by infrared spectroscopy（IR）,nuclear magnetic resonance hydrogen spectroscopy（¹H-NMR）and high-resolution mass spectrometry（MS）.The synthesized bifunctional ligands were then used for the modification of liposomes to prepare functional drug-loaded liposomes with curcumin（Cur）and dehydrocostus lactone（DL）as model drugs.The pharmacological properties such as particle size,polydispersity index（PDI）,zeta potential and morphological appearance of the liposomes were evaluated by Malvern laser nanoparticle sizer and transmission electron microscope（TEM）.The encapsulation rate of the liposomes was determined by periplasmic method.To examine the in vitro stability of liposomes and the changes in the reduced environment of simulated tumor cells.Finally,the cytological behavior of hepatocellular carcinoma cells Hep G2 was evaluated.Cytotoxicity was examined by MTT assay.The cellular uptake was also quantitatively and qualitatively analyzed by flow cytometry and inverted fluorescence microscopy.Results:The functional ligand molecule GA-SS-Chol was successfully synthesized,and the correct structure was verified by IR,¹H-NMR and MS.Modification of liposomes with the ligand GA-SS-NH₂confers active liver targeting and reduction responsiveness in hepatocellular carcinoma cells.After optimization of the preparation and prescription processes,the particle size of the resulting drug-loaded liposomes was uniformly about（143.6±2.86）nm,and the zeta potential could reach（-33.4±1.93）m V.The liposomes have with good stability and the encapsulation rate was about（84.3±2.1）%.The particle size of the liposomes became larger and the structure was destroyed under the DTT reduction environment.Finally,the in vitro anti-tumor activity and cellular uptake efficiency of the drug-loaded liposomes were investigated by cellular assays.The results showed that the modified liposomes had better hepatocarcinogenic effects than the normal liposomes and free drugs.The modified liposomes could be more and more effectively taken up by hepatocarcinoma cells,thus producing better efficacy against hepatocellular carcinoma.This study has great potential for the treatment of hepatocellular carcinoma and provides new dosage form ideas for the clinical use of anti-hepatocellular carcinoma drugs.