| Three negative breast cancer(TNBC)is a special type of breast cancer which is negative for estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor(HER2).It accounts for the 15%～20%of the total number of breast cancer.It has the characteristics of early onset,high pathological grade,high invasiveness,easy metastasis,poor prognosis and high mortality.Due to the lack of targets for endocrine and anti HER2 therapy,TNBC has no targeted standard treatment plan yet,and chemotherapy is still the main treatment.Adriamycin(DOX),a representative of anthracycline is an indispensable drug for TNBC chemotherapy.But,the DOX drug resistance often occurs,which severely limits its clinical application.Astragaloside is a natural compound extracted from Astragalus membranaceus,which has strong antioxidant activity.many studies have shown that astragaloside has a good antitoxic effect on adriamycin while a few reports have reported that astragaloside can sensitize the drug resistance of hepatoma cells.However,whether astragaloside can reverse or enhance the sensitivity of DOX in the treatment of breast cancer is rarely reported.Therefore,astragaloside has potential and advantages as sensitizer.Astragaloside is difficult to dissolve in water,but adriamycin hydrochloric acid is very water-soluble.How to achieve the common delivery of the two drugs and how to improve the targeting of the co delivery system are the key to the combination of two drugs and the preconditions for the combined use of two drugs to effectively reverse the drug resistance and to enhance the effect of the treatment of TNBC.So we started from the study of astragaloside reversing the drug resistance of TNBC adriamycin,and carried out a series of studies,including the construction of adriamycinastragaloside liposome common delivery system,synthesis of a new type of folic acid ligand FA-R8-cholesterol derivatives,the target evaluation of FA-R8 double modified liposomes in vitro and in vivo,construction of FA-R8 double modified doxorubicin astragaloside co delivery system and its effect on breast cancer in vivo and in vitro and a comprehensive predictive analysis of drug resistance mechanism of astragaloside reverses adriamycin.1.Reversal effect of astragaloside on doxorubicin resistance of TNBCIn this chapter,the effects of astragaloside and doxorubicin on the proliferation of MDA-MB-231 cells and MDA-MB-231/DOX cells were investigated by MTT test,and the synergistic effect of astragaloside on doxorubicin treatment and the reversal of chemotherapeutic drug resistance were investigated.The results showed that astragaloside has synergistic effect on adriamycin inhibiting the proliferation of MDA-MB-231 cells and MDA-MB-231/DOX cells.Using astragaloside solution of non-toxic concentration(3,1,0.5 mu ug/mL)to treat MDA-MB-231 parent cells,it was found that the concentration of astragaloside was 1～3ug/mL,which had a sensitizing effect on the proliferation of adriamycin to inhibit the proliferation of MDA-MB-231 parent cells,and the sensitization multiplier was about 1.6.Using astragaloside solution of non-toxic concentration(10、5、3、1、0.5 μg/mL)to treat MDA-MB-231/DOX cells,it was found that astragaloside could reduce the IC50 of,when the concentration of astragaloside was 3～10ug/mL,the reversal multiplier was about 3.The above results certificated that could reverse doxorubicin resistance in MDA-MB-231/DOX cells.In addition,We optimized the compatibility ratio between doxorubicin and astragaloside in vitro cell test,and the the best compatibility ratio was 1:3.2.The construction of LPs-DOX/AS co-delivery system.In this chapter,HPLC-UV method to determine the content of doxorubicin and HPLC-ELSD method to determine the content of astragaloside were established,which had prepared for follow-up studies.In this chapter,technology of liposome was used to constructe DOX-AS co-delivery system,and ethanol injection method combined with ammonium sulfate gradient method were adopt.Encapsulation rate,drug-loading rate,particle size and PDI value were selectived as the evaluation index,and single factor experiment was adopt to determine the suitable phospholipid species was S100,the removal method of free drugs was hyper-filtration,and the incubation temperature was 50℃.L9(34)orthogonal test was used to Optimize the preparation parameters of liposomes,and the evaluation index was the comprehensive score,which encapsulation rate and drug-loading rate each accounted for 50%.The results showed that the drug lipids had the greatest influence on encapsulation rate and drug-loading rate.The optimum preparation process condition:the phospholipid concentration was 8mg/mL,the ratio of phospholipid to cholesterol is 10:1 and the ratio of lipid to astragaloside was 5:1.Process verification shows that the liposome preparation method is stable and reliable.IR determination results proved that the drugs was successfully loaded in liposome co-delivery system,and the characterization results showed that particle size was 102.6±0.2nm and its distribution was uniform(PDI<0.3),Zeta potential was-11.2±1.35mV.3.The construction of FA-R8-LPs delivery system and its targeted evaluationWe took the EDC.HCL/HOBT/DMAP system as coupling agent to synthesize new ligand derivatives(FA-R8-cholesterol).MALDI-TOF-MS and IR identification results showed that the target product was successfully synthesised,and the purity of the product was greater than 80%,which were tested by TLC and HPLC.Cytotoxicity test results showed that FA-R8-cholesterol had no cytotoxic effect on HaCaT cells and MDA-MB-231 cells.The affinity between the new ligand and its receptor have still existed,which was tested by Elisa kit.Flow cytometry and laser confocal microscopy were used to evaluate target ability in vitro and the model drug is DOX.The results showed that cell intake of DOX in FA-R8-LPs group was the highest,and then R8-LPs group,and free drug group was lowest.The results also showed that the drug is located around the nucleus in cells,which were tested by laser confocal microscopy.Live imaging technology of small animals was adopt to evaluate vivo targeting effect and the model drug is DIR(a near infrared fluorescent dyes).The results showed that the FA-R8-LPs group had the strongest fluorescence in tumor tissues and predicted better tumor targeting.4.The construction of FA-R8-LPs-DOX/AS co-delivery system and pharmacodynamic evaluationIR determination results proved that the drugs was successfully loaded in FA-R8-LPs co-delivery system.The characterization results showed that particle size was about 109nmand its distribution was uniform(PDI<0.3),the encapsulation rate of doxorubicin and astragaloside respectively were about 98%and 95%,the drug-loading rate of doxorubicin and astragaloside respectively were about 4.5%and 14%.MTT test results in MDA-MB-231 cells displayed the IC50 of DOX group,LPs-DOX/AS gourp,R8-LPs-DOX/AS gourp and FA-R8-LPs-DOX/AS gourp respectively were 0.2776±0.0039 μmol/L,0.2388±0.0077 μmol/L,0.2163±0.0034 μmol/L and 0.1846±0.0083μmol/L,which followed that the IC50 of FA-R8-LPs-DOX/AS gourp was least.MTT test results in MDA-MB-231/DOX cells also showed that FA-R8-LPs-DOX/AS gourp had the least IC50,the IC50 was 0.3279±0.0045 μmol/L.DOX resistance in MDA-MB-231/DOX cells.The results cell apoptosis assay pointed out FA-R8-LPs-DOX/AS co-delivery system could effectively promote cell apoptosis.The results of in vitro test were further verified by vivo test.The tumor volume of FA-R8-LPs-DOX/AS gourp was significantly smaller than the control group and DOX group.After treatment 30 days,the TGI values from large to small were FA-R8-LPs-DOX/AS group,R8-LPs-DOX/AS gourp,LPs-DOX/AS gourp,LPs-DOX group and DOX group.The above results demonstrated that FA-R8-LPs-DOX/AS co-delivery system could effectively reverse DOX resistance and had good anti-tumor effect in treatment of TNBC.5.The mechanism research of Astragaloside-Ⅳ reversing DOX resistance in TNBC by transcriptomicsIn this chapter,transcriptomics was used to predictively analyze the mechanism of Astragaloside-Ⅳ reversing DOX resistance in TNBC.The results of bioinformatics analysis showed that there were 899 up-regulated genes and 1001 down-regulated genes had been identified between negative group and control group,and 748 up-regulated genes and 802 down-regulated genes had been identified between teatment group and control group.KEGG analysis showed that the mechanism of DOX resistance is mainly related to up-regulation of MAP3K14,BIRC3,TNC,LAMC3,CX3CL1 and other genes,which existed in NF-kappa B,PI3K-Akt,TNF,p53,etc.FA-R8-LPs-DOX/AS co-delivery system could effectively reverse DOX resistance in TNBC,which was mainly achieved by down-regulation of GPC4,SFRP5,FLT4,NFATC1,PAK1 and other genes by regulating such as Wnt signaling pathways,MAPK signaling pathways and Axon guidance. |
