| Objective:Paclitaxel(PTX)was encapsulated in amphiphilic targeted polypeptide vector P15(YIGSRHHHLLLGFLG)to obtain targeted drug delivery nanospheres P15 PTX.It was determined by cell experiments that P15-PTX had better advantages than PTX in inhibiting tumor cell proliferation,inducing tumor cell apoptosis and high cell uptake,and its main mechanism was determined by analyzing the changes of cell cycle and the expression of intracellular related proteins.To determine the therapeutic effect of P15-PTX in animal model,compared with the toxic and side effects and mechanism of PTX on various organs,in order to make characteristic work on the worldwide problem of cancer diagnosis and treatment and put forward new technologies and methods with potential application value.Methods:1.P15 was synthesized and purified by solid phase synthesis and high performance liquid chromatography.2.Optimize the synthesis conditions of P15;3.P15 paclitaxel loaded nanospheres P15-PTX was obtained by dialysis method,and the morphology,size and structure of P15-PTX were characterized,and the drug loading rate,entrapment efficiency and critical micelle concentration(CMC);4.To explore the drug release rule and release equation of P15-PTX in vitro by simulating different p H values(p H=1.2,p H=4.5,p H=6.8,p H=7.2).5.Taking human breast cancer cell line MCF-7 as the research object,the inhibition rate of tumor cell proliferation and the ability of inducing tumor cell apoptosis of P15-PTX were calculated by cell proliferation test and apoptosis test.6.Annexin V/PI double staining flow cytometry was used to detect the action cycle of PTX and P15-PTX.7.The cell uptake experiment was carried out by laser confocal microscope to observe the difference of P15-FITC-PTX and FITC-PTX uptake by MCF-7 cells.8.Western blotting was used to detect the expression of 67 LR protein,ERK1/2 protein,matrix metalloproteinase MMP-9 protein,bcl-2 protein and caspase-9 protein.9.The mouse tumor model was established by BALB/c tumor-bearing mice and 4T1 mice breast cancer cells,the tumor volume change curve was calculated,and the tumor mass comparison map was drawn.10.The tail vein blood and eyeball blood of mice were taken to detect the physiological and biochemical indexes;11.The tumors of each group were taken out and the changes of bcl-2 protein and caspase-9 protein content were analyzed.Results:1.The molecular weight of the peptide was 1719.996,which was in line with the molecular weight of the target product.After purification,the purity was about 94.08%.2.Controlling the variable resin type,feeding molar ratio and catalyst under the same other conditions,finally using 2-Cl resin,feeding molar ratio of 1:0.5 and catalyst is HBTU,the connection rate is the highest,that is,the connection effect between resin and amino acid is the best;3.P15 and PTX were mixed at an optimal ratio of 6:1 to form regular and round microspheres;P15-PTX composites are spherical,and their morphology and particle size are relatively uniform,with an average particle size of about 300-600 nm.According to the formula,the loading rate and encapsulation rate were 29.61% and 34.28%,respectively.CMC value is about 0.0032 g/L;4.In vitro drug release of P15-PTX was p H-dependent.In the buffer solution of p7.4,the cumulative drug release rate was 4.7843% after 2 h and 9.2465% after 80 h.In the buffer solution at p H6.8,the cumulative drug release rate was 7.5908% after 2 h and 19.3627% after80 h.In the buffer solution with p H4.5,the cumulative drug release rate was 12.8312% after 2h and 22.14569% after 80 h.In the buffer solution with p H1.2,the cumulative drug release at2 h was 13.9029%,and the cumulative drug release at 80 h was 25.0744%,which had the highest correlation with the Ritger-Peppas model in the drug release equation.5.The cell viability of PTX and P15-PTX at 25μg/m L was 72.2±4.8% and 53.5±3.55%,respectively.The survival rates were 46.9%±2.46% and 30.2%±1.91% at 400μg/m L,respectively.The cell inhibition rate of p15-PTX was 1.61 times and 1.31 times higher than that of PTX,respectively.The apoptosis rate of PTX was 11.7% at 50μg/m L,29.9% at200μg/m L,47.6% at 400μg/m L,and 29.2% at 50μg/m L,41.2% at 200μg/m L,and 67.3% at400μg/m L at the same concentration of PTX.Under the same paclitaxel content,the apoptosis rate of P15-PTX was 1.54 times higher than that of PTX on average.6.In G1 phase,the content of PTX and P15-PTX decreased,the content of S phase also decreased,and the content of G2 phase increased obviously.Among them,the content of P15-PTX G2 phase was 44.83%.The content of P15-PTX G2 phase was 18.86%.7.P15-FITC-PTX and FITC-PTX are mainly enriched in the cytoplasm,and the fluorescence intensity of P15-FITC-PTX is much stronger than that of FITC-PTX at 30 min and 8h.8.Under the condition of the same amount of β-actin,the expression of ERK1/2 protein and caspase9 protein increased significantly with the increase of drug concentration,while the expression of MMP-9 protein and bcl-2 protein decreased significantly with the increase of drug concentration.9.Compared with the PBS tumor-bearing control group,the tumor mass of PTX group and P15-PTX group decreased,and the therapeutic effect of P15-PTX group was more obvious.10.Compared with the blank control group and PBS control group,the levels of WBC,RBC,PLT,NEUT%,LYM%,TP and ALB in PTX group and P15-PTX group decreased,while ALT,ALP and Bun increased,and the rising and decreasing levels in PTX group were more obvious.11.In the case of the same amount of β-actin,the expression of caspase9 protein increased with the increase of drug concentration,while the expression of bcl-2 protein decreased with the increase of drug concentration.12.In the same time,the content of PTX entering the tumor was about 19.67%,which was92.78%,which was about 4.72 times higher than that of the same period last year.13.The observation results are discussed in the third part.Conclusion:1.The P15 peptide obtained by solid phase synthesis has good drug loading ability,and the prepared P15-PTX has the advantages of small particle size,regular morphology,easy dissolution,low toxicity,high targeting and good drug release ability.Part of the effect of 2.P15-PTX is to block the G＿2max M phase of tumor cells,has a good ability to inhibit the proliferation and uptake of tumor cells,and induces apoptosis by regulating the expression of bcl-2 and other proteins.3.P15-PTX has a good therapeutic effect on tumor-bearing mice,and its side effects are much lower than that of PTX,suggesting the potential of amphiphilic peptides for the construction of new drug carriers. |
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