Preparation Of Betulinic Acid Liposome And Its Targeting Effect On Hepatoma Cells In Vitro

Objective: To prepare hyaluronic acid(HA)modified betulinic acid liposome(HA-BA-L)and characterize it,to study its inhibitory effect on the proliferation of hepatoma cells and its targeting effect in vitro.Methods:1.Pre formulation study of betulinic acid liposome(BA-L): High performance liquid chromatography(HPLC)was established to detect the content of betulinic acid(BA),and the methodology of specificity,precision,repeatability and sample recovery were investigated;the oil-water partition coefficient of BA was determined by flask method;the entrapment efficiency of liposome was determined by low speed centrifugation method.2.Formulation optimization of BA-L:(1)BA-L was prepared by film dispersion method,ethanol injection method and active drug loading method.The best preparation method of BA-L was selected with encapsulation efficiency,particle size and PDI as evaluation indexes.(2)BA-L was prepared by the optimal method.The formulation of BA-L was optimized by single factor experiment and response surface design.The best liposome formulation was screened and validated.The particle size,PDI and potential of BA-L were measured by Malvern particle size analyzer,and the entrapment efficiency and drug loading of BA-L were determined by low speed centrifugation.(3)The stability of BA-L in PBS solution,serum and complete cell culture medium was investigated.The stability of BA-L at room temperature and 4℃ was also investigated.3.Preparation of HA-BA-L:(1)Based on the optimal formulation of BA-L,BA-L loaded with octadecylamine was prepared by ethanol injection method.HA was modified on the surface of BA-L loaded with octadecylamine by electrostatic binding principle to prepare HA-BA-L.the particle size,PDI and potential of HA-BA-L were investigated.The encapsulation efficiency and drug loading of HA-BA-L were determined.(2)The leakage rate of HA-BA-L at room temperature and 4℃ was investigated;the hemolysis of HA-BA-L was investigated by in vitro hemolysis test;the drug release characteristics of HA-BA-L in different p H release media were investigated by in vitro dialysis.4.Study on the anti hepatoma effect of HA-BA-L in vitro:(1)The inhibitory effects of BA,BA-L and HA-BA-L on the proliferation of LO2 human normal stem cells,Hep G2 and SMMC-7721 hepatoma cells in vitro were evaluated by MTT method,and the effects of liposome excipients on the activity of Hep G2 and SMMC-7721 hepatoma cells were also investigated.(2)The hepatoma cells were treated with BA,BA-L and HA-BA-L to observe the colony formation and migration of hepatoma cells.Meanwhile,the reactive oxygen species,total antioxidant capacity,lactate dehydrogenase(LDH)and asparagus in the culture medium were detected Aminotransferase(AST)activity.Objective to explore the anticancer effect of HA-BA-L by comparing the changes of BA,BA-L and HA-BA-L on hepatocellular carcinoma cells.5.The in vitro targeting effect and mechanism of HA-BA-L:(1)Rhodamine B(RHB)was used to load into liposomes,the uptake of targeted fluorescent liposomes(RHB-HA-L)and non targeted fluorescent liposomes(RHB-L)was detected by Hep G2 hepatocarcinoma cells under fluorescence microscope.(2)The uptake of RHB-L and RHB-HA-L by Hep G2 cells was evaluated by the fluorescence value of protein uptake per milligram.(3)The uptake of RHB-HA-L by Hep G2 hepatoma cells was observed under fluorescence microscope by blocking ha receptor with competitive inhibitor(free HA),and the uptake was detected by fluorescence spectrophotometry.(4)The expression of CD44 on the surface of Hep G2 hepatoma cells was observed by immunohistochemistry.The targeted effect of HA-BA-L on Hep G2 hepatocarcinoma cells was verified by the uptake rate of fluorescent liposomes.(5)After incubation of Hep G2 hepatoma cells with BA,BA-Land HA-BA-L,the expression of Rock1,IP3 and Ras protein in Hep G2 hepatoma cells were detected by(Western Blot,WB).The possible mechanism of HA-BA-L targeting treatment of Hep G2 hepatocarcinoma cells was analyzed.Result:1.Pre formulation study of BA-L: the content of BA was determined by HPLC.The linear relationship between BA concentration and peak area was good in the range of 10-320 μg/m L(r = 0.9999).The precision,repeatability and recovery of the instrument met the requirements of methodology.The log P value of oil-water partition coefficient of BA was 2.45±0.20,which was suitable for the preparation of liposomes.15 min low speed centrifugation 3000(r/ min)was used,the entrapment efficiency of BA liposomes could be effectively determined by the method.2.Formulation optimization of BA-L:(1)The encapsulation efficiency of BA-L prepared by film dispersion method,ethanol injection method and active drug loading method were 68.5%,75.3% and 47.9%,respectively;the particle sizes were 321.2 nm,175.6 nm and 279.7 nm,and the PDI were 0.55,0.15 and0.30,respectively.BA-L prepared by ethanol injection method has more advantages in encapsulation efficiency and particle size than film dispersion method and active drug loading method.(2)The optimal formulation and preparation process were obtained by response surface methodology.The optimal formulation and preparation process were determined as follows: the mass ratio of egg yolk lecithin to cholesterol was 10:1,the mass ratio of egg yolk lecithin to BA was 8:1,4 m L ethanol,8 m L phosphate buffer(PBS,p H7.4),and the preparation temperature was 53 ℃.The average entrapment efficiency of BA-L was 84.62%,the average drug loading was 10.70%,the average particle size was 135.7 nm,the average potential was-13.7 m V,and the average dispersion coefficient(PDI)was 0.22.(3)the particle size was stable in PBS solution,serum and complete cell culture medium,which were all less than 150 nm at 48 h;the stability was good when stored at 4℃ for 15 d.3.Preparation of HA-BA-L:(1)The average particle size of HA-BA-L was155.4 nm,PDI was 0.25,average potential was-18.2 m V,average entrapment efficiency was 83.13%,and average drug loading was 9.62%.(2)The stability of HA-BA-L was good after stored at 4℃ for 15 days;hemolysis test showed that HA-BA-L had no hemolysis phenomenon and good biocompatibility;the release effect of HA-BA-L in vitro was investigated.The release rate of HA-BA-L at p H 6.5 was better than that at p H 7.4,and there was no sudden release.4.Study on the anti hepatoma effect of HA-BA-L in vitro:(1)The results showed that the liposome excipients had no effect on the activity of Hep G2 and SMMC-7721 hepatoma cells;BA,BA-L and HA-BA-L had no inhibitory effect on the activity of LO2 normal hepatoma cells;the inhibitory effect of BA,BA-L and HA-BA-L on the proliferation of Hep G2 and SMMC-7721 hepatoma cells was concentration and time-dependent,and the inhibitory effect of HA-BA-L on the proliferation of Hep G2 and SMMC-7721 hepatoma cells was stronger than that of BA-L and BA.(2)BA,BA-L and HA-BA-L can inhibit the production of Hep G2 and SMMC-7721 hepatoma cell colonies,inhibit the migration of Hep G2 hepatoma cells,increase the activities of LDH and AST in the culture medium of Hep G2 hepatoma cells,and enhance the reactive oxygen species and total antioxidant capacity of Hep G2 hepatoma cells.5.The in vitro targeting effect and mechanism of HA-BA-L:(1)The fluorescence intensity of RHB-HA-L uptake by Hep G2 cells was significantly higher than that of RHB-L.(2)The uptake of RHB-HA-L by Hep G2 cells at 12 h and 24 h was 1.69 and 2.75 times higher than that of RHB-L,respectively.The results showed that RHB-HA-L with targeted fragments could actively target Hep G2 cells,and then significantly improve the uptake of RHB-HA-L by Hep G2 cells.(3)By adding a competitive inhibitor(free HA)to block ha receptor,the uptake of RHB-HA-L in Hep G2 hepatoma cells decreased,which further verified that RHB-HA-L could actively target ha receptor on Hep G2 hepatoma cells,indicating that RHB-HA-L has a targeting effect on hepatoma cells.(4)The expression of CD44 in Hep G2 hepatoma cells treated with BA,BA-L and HA-BA-L was compared.The results of immunohistochemistry showed that the expression of CD44 in Hep G2 hepatoma cells treated with HA-BA-L was lower than that in BA-L group.It may be that HA-BA-L was recognized and combined by CD44 receptors distributed on the surface of Hep G2 hepatoma cells,which confirmed the targeting effect of HA-BA-L.(5)WB results showed that BA,BA-L and HA-BA-L could down regulate the expression of Rock1,IP3 and Ras protein in Hep G2 hepatoma cells.Compared with BA and BA-L groups,HA-BA-L significantly down regulated the expression of Rock1,IP3 and Ras protein,suggesting that the down regulation of ROCK1-IP3-RAS in CD44 downstream signaling pathway is the expression of HA-BA-L targeted inhibition of Hep G2 hepatoma cell proliferation.Conclusion:1.In this study,BA-L was prepared by ethanol injection method.After loading octadecylamine,HA-BA-L was prepared by electrostatic binding principle.The particle size was 155.4 nm,the potential was-18.2 m V,the PDI was 0.25,and the encapsulation efficiency was 83.13%.2.HA-BA-L has obvious targeting effect on Hep G2 hepatoma cells.CD44 is an important protein of HA-BA-L.Its targeting effect may be to inhibit the proliferation of hepatoma cells through rock1-ip3-ras pathway,so as to play a targeted anti-liver cancer role.