| Breast cancer is a malignancy with high incidence rate and it is considered as a major threat to human health.China has the highest incidence of breast cancer in the world.The treatment methods for breast cancer mainly include surgery,chemotherapy radiotherapy and immunotherapy.As for the treatments of breast cancer,chemotherapy drugs in clinic(paclitaxel,doxorubicin and cisplatin)are only able to exert therapeutic effects by killing differentiated breast cancer cells effectively.And they are often of a substantial in vivo toxicity and cancers in patients receiving these drugs are easy to relapse after treatment,etc.Currently,it is widely recognized that breast cancer stem cells in tumor tissues will become resistant to chemotherapy after a period of time after a period of dormancy,which is the main cause of cancer relapse.Through self-renewal and differentiation to form new tumors,breast cancer cells in these patients would have the chance to recur or even develop metastasis.In view of the above problems,we developed three schemes.Firstly,we used two kinds of high-selective anti-tumor drugs which effectively kill breast cancer cells and stem cells,respectively.We could improve the treatment effects of breast cancer in this way.Secondly,we applied a series of drug combinations for breast cancer.Thirdly,we tried to load the drugs on nano-carriers,which could target the cancer cells effectively and reduce the toxicity effectively.In this study,we tested the combination of the above three schemes in combination with traditional anti-breast cancer drug docetaxel and new anti-breast cancer stem cell drugs salinomycin sodium for breast cancer.The two drugs are effective on breast cancer cells and breast cancer stem cells,respectively.The use of these two drugs may improve the synergistic killing of breast cancer and may enhance the therapeutic effects of breast cancer.Salinomycin is a kind of cyclic ether antibiotics,which can selectively act on a variety of tumor stem cells,including breast cancer stem cells and so on.According to reports,salinomycin sodium(SAL)also has a similar effect and it has a higher effect-price ratio.Docetaxel(DTX)is currently the chemotherapy option for breast cancer.PLGA[poly(lactic-co-glycolic acid)] is a kind of polymer medicinal material which was approved by the FDA.It has the characteristics of high biocompatibility,no toxicity,and so on.As a new type of non-toxic material,D-?-tocopherol acid poly-ethylene glycol succinate(TPGS)has enhanced MDR effect.It is also effective to suppress the efflux of P-glycoprotein and enhance the bioavailability of P-glycoprotein blocking drugs.Thus it can effectively overcome the multi-drug resistance of tumor cells.Accordingly,this paper applied nanoparticles with PLGA-TPGS as two kind of drug carrier,which were loaded with anti-breast cancer stem cell drugs SAL and anti-breast cancer drugs DTX targeting breast tumor sites to achieve effective treatment of breast cancer and substantial reduction in drug toxicity to normal body tissues.In this study,we established the determination method of HPLC in vitro for SAL and DTX.SAL's linear equation: A=1217.7C-3398(r2= 0.9999),the linear range was 6.25 ~ 1000 ?g/mL.DTX's linear equation: A=21836C+8557.8(r2= 0.9999),the linear range was 1~100 ?g/mL.The parameters used were in line with the requirement of drug content determination.HPLC method was established for the determination of entrapment efficiency,drug loading and in vitro release of PLGA nanoparticles for the next-step drug loading.We applied the MCF-7 breast cancer cell line as a model.With serum-free suspension culture technology,we selected MCF-7 microspheres(MCF-7-TS)for breast cancer stem cell model.We used breast cancer stem cell phenotypic marker antibody CD44+CD24-expression as a measure index(gold standards for breast cancer stem cells identification)and the breast cancer stem cell model MCF-7-TS were identified,the results showed that MCF-7-TS cells CD44+CD24-expression rate was over 60%.So we chose MCF-7-TS as breast cancer stem cell model for subsequent experiments.SAL of DTX had sensitizing effect in cytotoxicity test in vitro and in breast cancer cell lines.SAL of MCF-7-TS was chosen a specific and DTX to MCF-7-TS resistant and selective act on breast cancer MCF-7 cells.Finally,we screened MCF-7 and MCF-7-TS cells in breast cancer cell line with DTX and SAL with molar ratio of 2:1 and 1:1.We successfully prepared blank PLGA nanoparticles,nanoparticles loading salinomycin sodium(NS),nanoparticles loading docetaxel(ND)and nanoparticles loading salinomycin sodium and docetaxel(NSD)in accordance with by the proportion of total load with nanoprecipitation method.We optimized the prescription of PLGA nanoparticles.We selected the optimal ratio of PLGA and TPGS,the best dosing ratio of SAL and DTX which was used as the optimized PLGA nanoparticles formulation.Through the dynamic light particle size analyzer,transmission electron microscopy(TEM)of PLGA nanoparticles table structure was viewed.Particle size and zeta potential were characterized.The results showed that we successfully prepared uniformly distributed nanoparticles,which were particles in diameter of 70-100 nm in round shape and with zeta potential in-25 mV in the vicinity of the PLGA.We evaluated the in vitro release of NSD SAL and DTX under simulation of pH in normal blood and tumor tissue under conditions of media release,respectively.The results showed that the two drugs in NSD was with a certain degree of synchronous release effect within 10 days.We used Coumarin-6 as a fluorescence probe,loaded it into PLGA nanoparticles and to evaluate the cellular uptake of PLGA nanoparticles as a carrier in both of MCF-7 cells and MCF-7-TS cells.Compared with free Coumarin-6,cellular uptake of NP was both significantly observed in MCF-7 and MCF-7-TS cells.Moreover,we used DiR as a fluorescence probe,loaded it into NP to evaluate the distribution in organs and tumor by observed living images of tumor-loading nude mice.Compared with free DiR,cellular uptake of NP was both significantly observed in MCF-7 and MCF-7-TS cells,The result indicated NP have targeted property to both cells.Compared with free DiR,NP group could be mostly accumulated in tumors than other organs,indicated NP have targeted property to tumor tissue.Cytotoxicity-test of NP,free drugs and drug-loaded nanoparticles were investigated.The results showed that NP have no significant effect on MCF-7 and MCF-7-TS cells,drug-loaded nanoparticles can keep the activity of free drugs,NSD and NS+ND can keep the activity of free SAL and DTX,indicating that both of NSD and NS+ND have powerful cytotoxicity on breast cancer cells and stem cells.We investigated the pharmacokinetics of the three protocols SAL+DTX,NS+ND and NSD in male SD rats with HPLC-MS,respectively.The results showed that the SAL+DTX group was of the best in terms of sustained release of SAL.The NSD group was the best in terms of sustained release of DTX while the NSD group performed best in sustaining a preferred package loading ratio.The MCF-7 breast cancer nude mice model was successfully established.The toxicity and antitumor effect of the saline group,NP group,SAL group,DTX group,SAL+DTX group,NS group,group,ND group,NSD group,NS+ND group and the NSD group was evaluated.The results showed that there was no obvious toxicity of PLGA nanoparticles in nude mice.The antitumor activity was the best in the NSD group and no obvious systemic toxicity was observed.The tumor inhibition rate was 77.70 ± 4.92 in this group.Finally,in vivo anti-breast cancer stem cell activity was investigated by MCF-7-TS,which was isolated from tumor tissues in serum suspension culture.The results showed that NS+ND group and NSD group could significantly inhibit the number and volume of MCF-7-TS,indicating that the NS+ND group and the NSD group had a strong inhibitory effect on breast cancer stem cells.To sum up,the NSD can act on both breast cancer cells and breast cancer stem cells.Its anti-breast cancer activity is optimal in these test groups.NSD containing a synergistic ratio of two drugs can be an effective treatment in clinic for as a breast cancer treatment,which may be considered as a new treatment option. |
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