| The broad-spectrum anti-cancer drug docetaxel(DTX)was demonstrated to have significant efficacies on various cancers such as breast cancer,non-small cell lung cancer and ovarian cancer.However,the dose-dependence of the drug side effects of bone marrow suppression and allergic reactions limit its clinical use.In addition,docetaxel with poor biocompatibility,lack of specific targeting to tumor tissues and the preparations should be used immediately after preparation.Based on the above phenomenon,many researchers have tried to adopt new drug delivery systems such as nanoparticles,liposomes,nanomicelles and dendrimers to solve this problem.In our study,we designed and synthesized two-block polymer mPEG-PLA and two kinds pH-sensitive triblock polymer mPEG-PLA-PBAE.Docetaxel was loaded in the polymers to form the nanomicelles and the physicochemical properties,in vitro cytotoxic activity,in vivo anti-tumor activity and its anti-tumor mechanism were explored.The synthesis of the material:Two-block polymer mPEG-PLA(PELA)is formed by ring-opening method.After its end group is activated by acryloyl chloride,1-amino-4-methylpiperazine and 1.6-hexanediol diacrylate were added to form pH responsive triblock polymer mPEG-PLA-PBAE(PBAE1,PBAE2).The structure of each polymer and the molecular weight of the polymer was determined by 1H-NMR analysis.In order to evaluate the pH responsive behaviour of the polymer material,the alkaline dissociation constants pKb of each polymer were determined by acid-base titration method.Pyrene was selected as fluorescent probe to detect the critical micelle concentration of the polymers.The 1H-NMR spectrum shows the copolymers are well-joined and the molecular weights of the polymer PELA,PBAE1,PBAE2 are 4160Da,4945Da,5244Da,repectively.The pKb values of two PBAE polymers are all around 6.5,which is benefit for the drug release under weakly acidic conditions.And the critical micelle concentration(CMC)of the three polymer materials is 1.1?g·mL-1,3.?g·mL-1,0.8?g·mL-1 respectively,which indicating that the material more likely to form the stable micelle structure in aqueous solution.Film-hydration method is more convenient for the preparation of drug-loaded micelle.Therefore,in this study,film-hydration method was selected to prepare copolymer micelle and the laser particle size analyzer is used to measure the particle size distribution.The drug loading capacity and entrapment efficiency of the drug-loaded micelles were detected by high performance liquid chromatography.The stability of the micelles was carried out by observing the particle size changes of the micelles at room 25? and 37?,pH-responsive drug release behavior of the micelles were tested by dialysis method.The results show that the particle size distribution of docetaxel drug-loaded micelles was uniform and similar to the blank micelles,which was about 50nm.The drug loading capacity of different materials increased with more docetaxel added,and the drug-loading capacity of the materials was much the same,The maximum drug loading capacity of PBAE1 was 5.3%and the entrapment efficiency was reached to 93.8%.The particle size of the drug-loaded micelles PELA-DTX and PBAE1-DTX remained relatively stable at 25? for 48 h while the particle size of PBAE2-DTX changed greatly at 37?,48h,the stability deteriorated.PELA-DTX did not change the amount of release significantly under both conditions,whereas the release of docetaxel in PBAE1-DTX increased by 12.5%,which provided the possibility of release of docetaxel in weakly acidic tumor sites.In vitro cell experiments,Mouse Lewis lung cancer was chosed do the research.MTT assay was used to examine the effects of different docetaxel preparations on cell viability,flow cytometry method was used to detect the effect of the drugs on cell apoptosis and Western Blot experiment was used to further explore the mechanism of apoptosis in each group.The experimental results showed that the half-inhibitory concentration(IC50)of the free drug group,the non-pH-sensitive micelle group and the pH-sensitive micelle group showed a decreasing trend with the prolong of time,such as IC50(24h)>IC50(48h)>IC50(72h),indicating that various drugs have time-dependent toxicity effect on mouse Lewis lung cancer cells.In addition,experimental data showed that the pH-sensitive micelle PBAE1-DTX had stronger activity against Lewis lung cancer cells than the non-pH-sensitive micelle PELA-DTX and the free drug DTX.After treatment of Lewis cells for 48 hours,the apoptotic cells reached to 20.72%,29.71%,40.91%of free drug DTX,drug-loaded micelles PELA-DTX and PELA-PBAE1-DTX,respectively,which indicating that docetaxel preparations have effects on apoptosis of Lewis cells,while pH-sensitive PBAE1-DTX micelles have more advantages.Each drug group can increase the expression of pro-apoptosis protein Bax(DTX<DTX<PBAE1-DTX)and inhibits the expression of the anti-apoptosis protein Bcl-2(DTX>PELA-DTX>PBAE1-DTX),indicating that when comparing with free drug DTX,drug loaded micelle PELA-DTX and PBAE1-DTX more able to promote Bax expression and inhibit the expression of Bcl-2,then induce apoptosis of Lewis cells,among which pH-sensitive drug-loading micelles PBAE1-DTX with more advantages.Because docetaxel has the microtubule stablization behaviour,which can reduce the expression of ?3-tubulin(DTX>PELA-DTX>PBAE1-DTX),PBAE1-DTX is more effective in inhibiting the microtubule depolymerization and maintaining its structural stability.In conclusion,PBAE1-DTX can play a pro-apoptosis role as it can effectively regulate the expression of apoptosis proteins and ?3-tubulin.In in vivo animal experiments.HPLC was used to establish a method for determination of docetaxel in biological samples and the linearity,precision,and sample recovery were determined.In the experiment,C57BL/6 mice were selected as model animals to establish a subcutaneous vaccination model of Lewis lung cancer.After successful inoculation,the free drug DTX and drug-loaded micelles PELA-DTX,PBAE1-DTX were injected through caudal vein,and the plasma drug concentration was recorded over time.The distribution of docetaxel in organs at low dose of 10mg/kg or high dose of 20mg/kg were tested,and the change of volume of tumors and body weight of mice were recorded.HE staining can detect the morphology of tumor cell in the tumor tissue,then indirectly reflect the effect of the drug on the tumor.The experimental results showed that PBAE1-DTX micelle kept high plasma drug concentration over 24h after administration.At first 1 h after the administration of three docetaxel preparations,docetaxel were highly distributed in the heart,liver,spleen,lung,kidney and tumour tissues,the distribution of drugs in the tissues decreased after 3 hours,but the concentration of PBAE1-DTX in the tumor tissues was still highly at 24 hours compared with the other two groups.Compared with the saline group,each administration group was able to inhibit the growth of tumor volume,the PBAE1-DTX 20mg/kg group had the most significant effect.During the administration period,the body weight of the mice in each group did not change significantly.After HE staining,the morphology of cells in each group was observed,it was found that the tumor cells in the normal saline group were tightly arranged and the morphological structure was stable.However,the tumor cells were no longer compact and uniform in drug administration groups PBAE1-DTX and the cytoplasm was stained deep red color in large area,this shows that docetaxel has effects on tumor inhibition. |
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