Study On The Preparation Of Lupeol Liver Targeting Liposome And Its Anti-Hepatocarcinoma Effect

Objective:Hepatocellular carcinoma?HCC?is one of the most common malignant tumors in the world.Its morbidity ranks fifth in the world,and its mortality ranks third in the world.At present,there are many HCC treatment options,but the global 5-year survival rate of patients with liver cancer is still lower than 5%.The main reason is that the use of surgical resection and chemotherapy usually has serious side effects.Therefore,it is urgent to find and develop a new drug or drug delivery system to improve the above disadvantages,so as to achieve the purpose of prevention and treatment of HCC.Domestic and foreign studies have reported that the Chinese medicine Euphorbia fischeriana Steud,Euphorbiae Pekinensis Radix,Taraxacummongolicum Hand-Mazz have significant inhibitory effects on a variety of tumor models,and more and more studies show that the monomer components?triptolide and cantharidin?in Chinese medicines have a certain therapeutic effect on tumor diseases.Lupeol is the main chemical constituent of Euphorbia fischeriana Steud and Phyllanthus urinaria L.A large number of studies have confirmed that Lupeol has played a significant anti-tumor role in a variety of tumor cell lines and cancer models.However,due to the lack of water solubility and low bioavailability of Lupeol,its development and use are limited.Therefore,a new drug carrier is needed to improve its properties and targeting.In this study,we first constructed a Lupeol liposome with the ability of liver targeting and long circulation,and then studied its targeting and tumor related protein signaling pathway in HepG2 cells.Finally,SB?Sleeping Beauty?-mediated high-pressure tail vein injection was used to transfect AKT/c-Met-induced mouse HCC models as experimental animal models for further verify the antitumor ability,targeting ability and molecular mechanism of Gal-Lupeol-L under optimal dosage.This project provides a new idea for the study of Lupeol drug delivery system,and also provides a new basis for the pharmacodynamic evaluation of mouse model of liver cancer established by gene transfection in vivo.Method:1.HPLC was used to establish the method for the determination of Lupeol content;ultrafiltration centrifugation,membrane filtration and protamine precipitation were used to select the method for the determination of the EE of Lupeol liposomes;the EE was used as the evaluation index to investigate the preparation method of Lupeol liposomes;the single factor test was used to screen the factors affecting the Lupeol-L to provide a reference for the preparation of Gal-Lupeol-L.2.With the EE and particle size as the evaluation index,the orthogonal test was used to investigate the influence of the main influencing factors on the formation process of Gal-Lupeol-L and optimize the best formation process;the influence of different freeze-drying protectors on the EE and particle size of Lupeol liposomes was investigated and the best freeze-drying protectors were optimized;the morphology of liposomes was observed by TEM and SEM,the dynamic light scattering method was used to measure the particle size,PDI and zeta potential;the release behavior of free Lupeol,Lupin-L and Gal-Lupeol-L were investigated by dialysis method,and the simulated equation was fitted to the release behavior in vitro;the dilution stability and long-term stability of free Lupeol,Lupeol-L and Gal-Lupeol-L were investigated with the EE and particle size.3.In vitro targeted study,flow cytometry was used to detect the uptake effect of different dosage forms of Lupeol in HepG2 cells for qualitative study;HPLC was used to detect the uptake of the same dosage forms of Lupeol in HepG2 cells for quantitative study;in vivo targeting studies,the distribution of different organs of the same dosage and different dosage forms in different organs of FVB mice was measured using a live imager.4.HPLC was used to detect the concentration of Lupeol in different time points of SD rats with the same dosage and different dosage forms.The average concentration time data were analyzed by using DAS3.0 software and non atrioventricular model.5.In vitro pharmacodynamic evaluation,CCK8 method was used to determine the effect of the same dosage and different dosage of Luepol preparation on the survival rate of HepG2 cells;flow cytometry was used to detect the effect of the same dosage and different dosage of Luepol preparation on cell apoptosis and cycle;the expression of Akt/mTOR and RAS/MAPK signaling pathway related proteins in HepG2 cells was detected by western blot.6.Using high-pressure tail vein transfection technology to simultaneously overexpress AKT?with HA-Tag?and c-Met?with V5-Tag?in the liver of FVB/N mice as liver cancer model,study the same dose different dosage form Lupeol preparations on liver appearance,liver weight,and pathological tissue of AKT/c-Met-induced mice;qPCR was used to detect the markers of liver cancer with the same dose different dosage Lupeol preparations on AKT/c-Met-induced HCC mice;western blot was used to test protein expression about AKT/mTOR and Ras/MAPK signaling pathway after the different dosage forms of Lupeol preparations actted on AKT/c-Met-induced mice.Result:1.HPLC method for the determination of Lupeol was established;the solubility of Lupeol in chloroform was the highest,and it was insoluble in water;the membrane method was finally determined to be the method for the determination of the EE of Lupeol liposomes,and the membrane dispersion method was the method for the preparation of Lupeol liposomes;the single factor test method was used to determine that the biggest factors affecting the shaping process of Lupeol liposomes were drug lipid ratio and phosphorus bile ratio,PEG2000-DSPE.2.Through the orthogonal test process investigation,the EE is the index of investigation.Finally,the optimal molding process for Gal-Lupeol-L was determined to be 7:1 phosphorobiliary ratio,1:10 drug-lipid ratio,the mass percentage of Gal-PEG-DSPE in the prescription is 10%;the EE and particle size were used as evaluation indicators,and when sucrose is selected as the lyophilized protective agent,all proportions of particle size and EE are the qualified range.At the percentage of 15%,the EE was the highest,so it was finally determined that sucrose with a prescription weight ratio of 15%was a lyophilized protective agent;in the release degree examination,Free Lupeol released nearly 70%within 10h,and Lupeol-L?Gal-Lupeol-L released only about 40%within 10 hours.After the kinetic fit,it was found that the release of Free Lupeol was first-order kinetics equation,the release of Lupeol-L and Gal-Lupeol-L was the Weibull equation;the results of the dilution stability investigation show that Lupeol liposomes were relatively stable at a dilution of 20 times,and the results of long-term stability inspection indicated that Lupeol liposomes are stable in a refrigerator at 4?for 6 months.Free Lupeol results showed a slight hemolysis,while Lupeol-L,Gal-Lupeol-L is not hemolytic.3.The results of targeted qualitative study in vitro showed that the uptake intensity of targeted group was higher than that of free drug group and non targeted drug group.The results of quantitative study showed that Gal-Lupeol-L was 1.65 times of free Lupeol uptake,1.17 times of Lupeol-L uptake.The results of targeted studies in vivo showed that the fluorescence in the liver parts of the Gal-NR-L group continued to 24 h,while the fluorescence in the liver parts of the free NR and NR-L groups disappeared at 5 h..4.The results of pharmacokinetic study showed that the area under the drug time curve of the targeted group was about 3 times than the free drug group;the mean residence time MRT and plasma half-life T_1/2/2 of the targeted group were about 2.36 times and 3.21 times than the free drug group,respectively.5.The IC50 values of Free Lupeol,Lupeol-L,Gal-Lupeol-L on HepG2cells for 24 h and 48 h were 163?M,126?M,87?M;122?M,82?M,61?M.The apoptotic rates of free Lupeol,Lupeol-L and Gal-Lupeol-L were 4.3%,8.73%and 18.51%,respectively.Gal-Lupeol-L could block HepG2 cells in G2/M phase.The expression of Akt/mTOR related protein?p-akt308,p-akt473,p-rps6,FASN?and the main effector downstream of Ras/MAPK pathway p-ERK1/2,as well as the tumor proliferation marker PCNA in HepG2 cells were also decreased more obviously in Gal-Lupeol-L group than in free Lupeol group.6.HCC model co-expressed with AKT and c-Met plasmids transfected with high-pressure tail vein was successfully prepared.The liver index and liver weight of mice in the Gal-Lupeol-L administration group were significantly reduced compared with the free group?P<0.05?;Compared with free Lupeol group,the liver-lobular structure of the liver in the Gal-Lupeol-L group is clearer,vacuoles are more pronounced,and the cytoplasm is more abundant.Compared with the Free Lupeol and Lupeol-L groups,the mRNA expression levels of AFP,GPC3,and Epcam in the liver of mice were significantly reduced after Gal-Lupeol-L administration?P<0.01?;Compared with the free Lupeol group,the Akt/mTOR related protein?p-akt308,p-akt473,p-rps6,FASN?,the expression of p-ERK1/2 and the expression of PCNA,the tumor proliferation marker,decreased significantly in the Gal-Lupeol-L group.Conclusion:The EE is high and the particle size was less than 200.Compared with the free and non targeted liposomes,Gal-Lupeol-L had better targeting in vitro and in vivo;in pharmacokinetics,the average retention time MRT and plasma half-life T_1/2/2 of the liver targeted liposomes in vivo are better than those of the free liposomes;Gal-Lupeol-L show better cytotoxicity and apoptotic efficiency on HepG2 cells in vitro,and had a better effect on reducing expression of AKT/c-Met pathway-related proteins;the inhibition effect of Gal-Lupeol-L on Akt/c-met-induced HCC mice related pathway was better than that of free Lupeol and non targeting liposomes of Lupeol.