Experimental Study On The Activity Of Doxorubicin Loaded With Graphene On Breast Cancer

Objectives To establish the model of human breast cancer MCF-7 cell line in nude mice,to explore the effect of oxidized graphene loaded doxorubicin on the anti-tumor effect in nude mice and to alleviate the liver injury caused by chemotherapy.Methods Human breast cancer MCF-7 cells were cultured under normal conditions,and subcutaneously implanted single cell suspension was performed to construct a subcutaneous tumor model of human breast cancer MCF-7 cell line in nude mice.The 60 successfully modeled nude mice were randomly divided into 3 groups: saline control group,doxorubicin group,graphene loaded doxorubicin group,once every 2 days,a total of 28 days of intervention.The body weight was measured before each dose,and the nude mice were observed during the intervention.After the administration,the anesthesia was conducted,sample was taken and all nude mice were killed.The tumor inhibition rate of each group was observed,the specimens of subcutaneous xenografts were observed by HE staining for pathological examination to observe the morphology of the cells.The expression of ICAM and mmp2 were detected by immunohistochemistry.Blood samples were taken from nude mice to measure alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and liver tissue homogenate was used to measure SOD,GSH and other indexes.Data were analyzed by SPSS 17.0 and were expressed as mean ± standard deviation(x ± s).A single factor analysis of variance was used to compare the mean of multiple groups.The homogeneity of variance between two groups was tested by LSD,on the other hand the heterogeneity of variance was analyzed by Dunnett's T3 test.P <0.05 was considered statistically significant.Results Chapter 1: After subcutaneous injection of breast cancer MCF-7 cells single cell suspension,about 9 days later,we can see subcutaneous tumor nodules uplift,irregular shape,clear boundary,and hard with poor mobility.The inhibition rate of doxorubicin group and graphene loaded doxorubicin group was 22.62% and 31.48% respectively.There was significant difference between the saline group and the doxorubicin group as well as the graphene loaded doxorubicin group(P <0.05).The results of HE staining showed that the tumor cells in the saline group were closely arranged,cell volume increased and the shape was irregular.Tumor tissues of Doxorubicin group and graphene loaded doxorubicin group showed different degree of cell necrosis,cell structure disappeared and the necrotic area was homogeneous red dye.Immunohistochemistry showed that caspase-8 was expressed in the cytoplasm of breast cancer cells.Brown granules were found in the cells after the staining and they were distributed as crumbs or patches.Compared with the normal saline group,the expression of caspase-8 in the doxorubicin loaded group and the graphene loaded doxorubicin group increased significantly.The difference was statistically significant(P < 0.05).The expression of the graphene loaded doxorubicin group was higher than that of the doxorubicin group,and the difference was statistically significant(P < 0.05).The expression of PCNA protein was in the cytoplasm and part of cell membrane,the positive expression products showed brownish yellow after dyeing.Compared with the normal saline group,the expression level of doxorubicin group and graphene loaded doxorubicin group decreased,and the difference was statistically significant(P < 0.05).The expression of the graphene loaded doxorubicin group was lower than that of the doxorubicin group,and the difference was statistically significant(P < 0.05).Chapter 2: Graphene loaded doxorubicin group significantly reduced the levels of ALT,AST and ALP caused by doxorubicin-induced liver injury(P <0.05).Compared with the saline group,the level of MDA in the doxorubicin group and the graphene loaded doxorubicin group was significantly increased,and the level of GSH and SOD was significantly decreased(P <0.05).Compared with the MDA level in the doxorubicin group,the graphene loaded doxorubicin group decreased by 28%(P <0.05).Compared with the GSH level in the doxorubicin group,the graphene loaded doxorubicin group increased by 29.83%(P <0.05).Compared with the SOD level in the doxorubicin group,the graphene loaded doxorubicin group increased by 25.37%(P <0.05).The HE staining results of hepatic tissue showed that the structure of hepatic lobules was clear and complete in the liver tissue of nude mice in saline group,and the hepatic cords were arranged radially in the central vein of lobular vein.The microscopic liver cells of doxorubicin group showed swelling,degeneration,loose cytoplasm and unclear structure of hepatic sinusoid.The degree of liver cell degeneration in grapevine loaded doxorubicin group was relatively lower than that of the doxorubicin group,and there's no significant steatosis.Conclusions 1.Graphene loaded doxorubicin had significant inhibitory effect on the growth of subcutaneous transplanted tumors,the mechanism of it perhaps graphene loaded doxorubicin has up-regulated the expression of caspase8 and down-regulated PCNA protein in transplanted tissue.2.Graphene loaded doxorubicin can increase the level of GSH,SOD in the liver,reduce the level of MDA and ameliorate the liver injury caused by chemotherapy,suggesting that its action mechanism may be related to the better tissue permeability and drug utilization rate of graphene.