The Anti-liver Cancer Activity Of Salinomycin Loaded PEG-creamide Nanomicelles

We successfully designed salinomycin loaded PEG-creamide nanomicelles(SCM),which can both kill HepG2 cells and HepG2-TS.Because of the improvement of salinomycin water-solubility and the enhancement of carrier cancer-killing effects,coupled with the permeability of the micelles,more and more salinomycin was targetd to tumor,which provided a new strategy for the treatment of liver cancer.Firstly,we established salinomycin detection methods by high performance liquid chromatography and liquid chromatogram/mass spectrum instruments,which provided the basis for the determination of SAL in vitro and in vivo.This method gave the results that when salinomycin concentration ranged from 7.82 to 1000 ?g/mL,its peak area showed a good linear relationship and the linear regression equation is A=1012.4C+7115.6(R2=0.9999).Under this condition,the salinomycin determination method was specific and the precision and accuracy of the method were both meet the requirements of the experiment within 3%,which indicated that the method could be used to determine the concentration of SAL in vitro.We also established the determination of salinomycin in vivo with fenofibrate as internal standard,got the linear regression equation A=0.005995C-0.01851(R2=0.999).The linear range of the method was from 20 to 1000 ng/m L,and the lower limit of quantification was 20ng/m L.When S/N?3,the limit of SAL detection was 4ng/mL,which can meet the experimental requirements.Secondly,HepG2 cells and the microspheres with stem cells(HepG2-TS)were cultured.The HepG2-TS were isolated from the HepG2 cells and cultured with the DMEM medium without serum.We investigated the interaction between SAL and PEG-ceramide in HepG2 cells,following the steps that mixturing the two free drugs with 1:2,1:3 and 1:4 molar ratios,then using them to kill HepG2 cells,and finally we got the conclusion that 1:2 and 1:4 were the good molar ratios for killing HepG2 cells.In order to choose the best synergistic ratio for the following experiments,we based on the two ratios to make the salinomycin loaded PEG-creamide nanomicelles respectively,and found that the ratio of 1:4 was good to choose.Thirdly,blank poly(ethylene glycol)-ceramide micelles and the optimal molar ratio(SAL:PEG-ceramide =1:4)of drug loaded PEG-ceramide micelles were prepared by thin film dispersion method.The particle size of obtained micelles was 11.02±2.34 nm,Zeta potential was-5.66±0.77,the encapsulation efficiency and drug loading were 76.74±4.05%,6.32±0.57%,respectively,and the accumulative release rate of 48 h was 65% when pH=7.4.Salinomycin loaded PEG-ceramide micelles were observed by transmission electron microscope,and the results illustrated that the micelles size was uniform,dispersed homogeneously without adhesion.Fourthly,we tested the killing effect of the drug-loaded micelles on HepG2 cells and HepG2-TS with optimal synergistic ratio.CCK-8 method was used to investigate the cytotoxicity of HepG2 cells and HepG2-TS.The Annexin V-FITC/PI double staining method was used to investigate the cell apoptosis.Colony forming test and mammosphere formation rate experiment were used to investigate tumor cells proliferation ability.Laser scanning confocal microscopy was used to study the tumor cells uptake ability of loaded fluorescent micelles.The results showed that SAL loaded PEG-ceramide micells could significantly improve the killing effect on HepG2 cells and Hep G2-TS,and enhance the ability of inducing apoptosis and inhibiting cell proliferation.Moreover,the micelles uptake ability for HepG2 cells and HepG2 microspheres was stronger than that of control group,which showed that PEG-ceramide micelles had better targeting characteristics to HepG2 cells and HepG2-TS.Fifthly,we gave a safety evaluation of the micelles carrier.We took tail vein injection,using its heart,liver,spleen,lung,kidney to do the CD68 immunohistochemisty experiment and HE staining,and did blood analysis.The results showed that PEG-ceramide micelles carrier almost haa no obvious toxicity to BALB/C mice,only with a slight toxicity of the liver and kidney.Second,we established the HepG2 tumor bearing nude mouse model and prepared the Dir loaded PEG-ceramide micelles to evaluate the micelles distribution and tumor targeting in animals.The results showed that the micelles were mainly distributed in liver and tumor tissue,having strong tumor targeting and tumor permeability.Prepared tumor frozen section,putting it in the laser confocal microscope to observe its tumor targeting characteristic,the result showed that the PECF loaded PEG-ceramide micelles have the maximum fluorescence intensity in mice tumor model,and showed the best targeting characteristics to tumor.LC-MS was used to analyze the PEG-ceramide micelles pharmacokinetic characteristics for the four groups of drugs,including PBS,free SAL,DSPE-PEG micelles and SAL loaded PEG-ceramide micelles.Results showed that the two drug loaded micelles especially PEG-ceramide micelles could reduce SAL release in plasma.Futhermore,the antitumor activity and systemic toxicity of the micelles were investigated from the size of the tumor,the weight of the tumor,the growth curve of the tumor volume and the change of the mice weight.Results indicated that the drug loaded PEG-ceramide micelles had no obvious systemic toxicity,and showed the best antitumor activity.In summary,SAL loaded PEG-ceramide micelles have a good inhibition for common liver cancer cells and liver cancer stem cells,showing a good application prospect.