| In recent years,the incidence and mortality of breast cancer have continued to rise,which was one of the leading causes of death among women around the world.According to the molecular classification of breast cancer,triple-negative breast cancer has the strongest invasiveness,the worst prognosis,and there is no effective targeted treatment,so it is prone to metastasis.Silybin has been known to inhibit breast cancer cell proliferation,induce breast cancer cell apoptosis and inhibit breast cancer metastasis through a variety of mechanisms,showing potential value in the treatment of invasive breast cancer.However,the poor water solubility and low bioavailability of silybin hinder its efficacy.In this study,a nano-drug delivery system will be designed to solve the above problems according to the complex characteristics of tumor microenvironment.The main research contents are as follows:The initiator was synthesized by substitution reaction,and the amphiphilic block copolymer poly(2-ethyl-2-oxazoline)-poly(lactic acid)was obtained by two ringopening polymerization,after acrylylation,Michael addition reaction took place with diacrylate and amine monomers,and the final product synthesized poly(2-ethyl-2oxazoline)-poly(lactic acid)-SS-poly(β-amino ester)triblock copolymer with pH response and glutathione response in tumor microenvironment.The structure of the copolymer was confirmed by 1H-NMR and its molecular weight was calculated.The critical micelle concentration(CMC)of the copolymer was determined by pyrene fluorescence probe.The particle size and Zeta potential of the blank micelle were measured by dynamic light scattering.The stability of the blank micelle was investigated by the change of particle size at different temperatures.The morphology of the blank micelle was observed by transmission electron microscope.And the safety of the blank micelle was investigated by hemolysis experiment.The results showed that the structure of the synthesized product was confirmed.The molecular weight of PEOz-PLA-SS-PBAE copolymer was 4741 Da.The critical micelle concentrations of PEOz-PLA and PEOz-PLA-SS-PBAE were 1.2 μg·mL-1 and 1.8μg·mL-1,respectively.The particle size of PEOz-PLA blank micelles was(69.25± 1.09)nm,PDI(0.260±0.003).The particle size of PEOz-PLA-SS-PBAE blank micelles was(155.30 ± 1.80)nm,PDI(0.168±0.004),the particle size distribution was uniform,and the morphology of the two micelles were spherical or quasispherical under transmission electron microscope,the size was uniform and the dispersion was good.The two kinds of blank micelles had good storage stability at 4℃;the hemolysis rate of the blank micelles of PEOz-PLA and PEOz-PLA-SS-PBAE materials was less than 5%,which proved that the prepared copolymer materials had good blood compatibility.Silybin micelles were prepared by hydration film method.A method for the determination of silybin content was established by high performance liquid chromatography(HPLC).The drug loading and encapsulation efficacy of silybin micelles were determined.The particle size and Zeta potential of drug-loaded micelles were measured by dynamic light scattering.The stability of drug-loaded micelles was investigated at different temperatures.The morphology of drug-loaded micelles was observed by transmission electron microscope.The release properties of silybin nanomicelles in different media were investigated.The results showed that silybin had a good linear relationship with the peak area in the concentration range of 6.25-200μg·mL-1,and the precision and recovery were in accordance with the regulations.The drug loading and encapsulation efficacy of PEOz-PLA micelles were(7.03±0.10)%and(89.55±1.29)%,The drug loading and encapsulation efficacy of PEOz-PLA-SSPBAE micelles were(5.48±0.04)%and(68.52±0.48)%.The particle size of PEOzPLA drug-loaded micelles was(73.91±0.53)nm,PDI(0.262±0.005).The particle size of PEOz-PLA-SS-PBAE drug-loaded micelles was(151.30±1.41)nm,PDI(0.174±0.021),the particle size distribution was uniform.Under transmission electron microscope,it was observed that the two micelles were spherical or quasi-spherical and uniformly distributed.The two kinds of drug-loaded micelles tended to decrease at 37℃ within 72 h,and were more stable at 4℃.Under the condition of pH6.0,the release rate of PEOz-PLA drug-loaded micelles was(43.90±1.24)%within 72 h,which was significantly higher than that of pH7.4(36.74±0.21)%.The release of PEOz-PLA drug-loaded micelles was basically the same with or without GSH.The release rate of PEOz-PLA-SS-PBAE drug-loaded micelles in pH6.0 was(44.46±2.26)%within 72 h,which was significantly higher than that in pH7.4(35.38±0.64)%.The release of PEOz-PLA-SS-PBAE micelles increased significantly when GSH was added under two kinds of pH conditions,and the maximum release was(64.54±0.67)%under pH6.0 and GSH conditions,which proved that the prepared micelles had the response of tumor microenvironment pH and GSH.Taking triple negative breast cancer MDA-MB-231 cells as the main object of study,the inhibitory effects of blank micelles,free drugs and drug loaded micelles on MDA-MB-231 cells were investigated by cytotoxicity test.The apoptosis induced by free drugs and drug-loaded micelles was investigated by cell flow assay,and the inhibition of MDA-MB-231 cell migration by free drugs and drug-loaded micelles was investigated by wounding healing assay.The ability of different silybin preparations to inhibit the invasion of MDA-MB-231 cells was investigated in Transwell cell invasion assay.The experimental results showed that different concentrations of blank micelles acted on cells,and the cell survival rate was more than 80%within 72 h,which proved that the material had good safety.The cytotoxicity test of drug-loaded micelles showed that PEOz-PLA-SS-PBAE-loaded micelles had a significant inhibitory effect on MDA-MB-231 cells.After treated with free drug,PEOz-PLA drug-loaded micelle and PEOz-PLA-SS-PBAE drug-loaded micelle for 24 h,the apoptosis rates of MDA-MB-231 cells were 7.6%,18.6%and 31.3%,respectively,indicating that PEOz-PLA-SS-PBAE drug-loaded micelles could significantly induce apoptosis of MDA-MB-231 cells.The migration rates of MDAMB-231 cells were 23.9%,15.5%and 10.4%respectively,indicating that PEOz-PLASS-PBAE drug-loaded micelles could significantly inhibit the migration of MDAMB-231 cells.The results of Transwell invasion test showed that PEOz-PLA-SSPBAE drug-loaded micelles could significantly inhibit the invasion of MDA-MB-231 cells by calculating the average number of cells per visual field after 24 hours of treatment with different silybin preparations.Therefore,the PEOz-PLA-SS-PBAE drug-loaded micelles prepared in this study have good inhibitory effect on tumor growth and anti-tumor metastasis in vitro,which lays the foundation for the further application of silybin. |
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