Preparation And Anti-tumor Effect Of Polymeric Micelles Loading With Ursolic Acid

Objective:The purposes of this project are to prepare ursolic acid-polymeric micelles(UA-PMs),study its physical and chemical properties,establish a method for determining the content of ursolic acid(UA)and evaluate its antitumor activity on Hepatic Carcinoma in vitro and in vivo.Methods:Firstly,HPLC method was used to determine UA and investigate the encapsulation efficiency,drug loading of UA encapsulated in micelles and the release behavior of UA-PMs in vitro.Secondly,UA-PMs were prepared by thin-film dispersion method and its size distribution,polydispersity index(PDI)and Zeta potential were determined by laser scattering technique.Moreover,pyrene fluorescence probe method was used in measuring the criticalmicelleconcentration(CMC)of empty PMs and UA-PMs.Thirdly,hepatocellular carcinoma cells HepG2 were cultured in vitro and treated with different concentrations of UA and UA-PMs with the 5-Fu as positive control,respectively.The ability changing in cell proliferation and metastasis were detected by MTT and wound healing assay,respectively.Fourth,KM mice were chosen for the acute toxicity experiment.With normal saline(NS)group as the negative control and 5-Fu as the positive control,the blank PMs,UA and UA-PMs were given by excessive dose in the acute toxicity test.To estimate the damage of drugs to the liver and kidney function,we collected randomly serum samples on days 0,3,7 and detected the biochemical indexes.Finally,heart,liver,spleen,lung and kidney were collected and stained with H&Estaining.Fifth,KM mice were chosen for the pharmacodynamics study.Then,hepatocarcinoma cell line H22 was inoculated into the abdominal cavity of mice.After a week,the peritoneal cells were collected,diluted to the appropriate concentration,and injected subcutaneously into the right hind leg of KM mice.The negative control group and the positive control group were the normal saline and 5-Fu,respectively,at the same time,the blank PMs,UA and UA-PMs were given by intraperitoneal injection at the appropriate time.In the meantime,to investigate the effect of UA-PMs on tumor growth,the mice were observed and the survival time of mice was recorded.The tumor inhibition rate and survival rate were calculated.Results:A method for determing the content of ursolic acid was successfully established and the standard curve is A=3.868C-1.610,R~2=1.000.The encapsulation efficiency and drug loading of UA-PMs are 59.85±1.68%and 5.44±0.47%,respectively.In vitro release test,compared with UA solution,UA-PMs did not have obvious burst release phenomenon in buffer solution(pH 7.4).The particle size and zeta potential of UA-PMs were 29.35±0.38nm and-0.75±1.30 mV with a PDI of 0.299±0.005.The critical micelle concentrations of blank PMs and UA-PMs were5.1×10~-33 and 2.3×10~-33 mg/mL,respectively.MTT assay showed that UA-PMs could inhibit significantly the proliferation of HepG2 cells,and the scratch assay demonstrated that UA-PMs did obviously inhibit the migration of HepG2cells.Moreover,both of the two experiments showed the concentration and time dependence.The results obtained from the organ coefficientmeasurement indicated that the positive control group(5-Fu)had toxic effect on the heart,the high dose of UA showed slight damage on the liver,and the spleen coefficient of high doses of UA and UA-PMs group were slightly higher.The biochemical test results showed that the indexes of AST,ALT in liver of high dose UA were slightly abnormal,other groups of drugs had no obvious effect on liver and kidney index.Meanwhile,the results showed that high dose of UA had slight damage on the liver by pathological section.Finally,pharmacodynamic experimental results illustrated that 5-Fu,UA and UA-PMs could significantly inhibit the growth of tumor in mice.Conclusion:UA-PMs showed the sustained release effect in vitro,and could inhibit the proliferation and migration of hepatocellular carcinoma cells in vitro.Acute toxicity test showed that UA and UA-PMs had no significant toxic effects on the heart,spleen,lung and kidney while the high dose of UA showed a slight damage on the liver.UA-PMs did play the role of inhibition on tumor growth.