| Part 1To create the drug sustained-release materials with nanofibrous structure loaded with gemcitabine and cisplatin by electrospinning technology and to investigate its function on drug release system in vitro.Objective:To create the drug sustained-release materials with nanofibrous structure loaded with gemcitabine(GEM)and cisplatin(CDDP)by electrospinning technology and to investigate its function on drug release system in vitro.Methods:With classical electrospinning technology,the blank electrospinning was contructed with polylactic acid(PLA)as raw material and hexafluoroisopropanol as solvent.Then the pharmacodynamic curves of gemcitabine and cisplatin were drawn with the MTT experiment of mouse bladder cancer cell line MB49,and according to the pharmacological principle,the optimal dose ratio of gemcitabine and cisplatin was calculated.Furthermore,according to the optimal drug dose ratio,the drug sustained-release materials with nanofibrous structure of gemcitabine hydrochloride and cisplatin were prepared by polylactic acid,gemcitabine and cisplatin as raw materials and hexafluoroisopropanol and dimethylformamide as solvents.The diameter distribution and characterization of electrospun nanofibrous materials were observed by scanning electron microscope(SEM).The drug sustained-release materials with nanofibrous structure with certain quality were completely dissolved by chloroform,and then the quality of gemcitabine in chloroform solution was detected by extraction technology and high performance liquid chromatography(HPLC)in order to calculate the load drug rate of above materials.The drug sustained-release materials with nanofibrous structure were put into 0.9%sodium chloride solution,which can simulate tissue fluid,and a small amount of solution was taken as samples at different time points.The concentration of gemcitabine in the sample solution at different time points was detected by high performance liquid chromatography(HPLC).According to the detection results,the drug release curve of the drug sustained-release materials was drawn.Results:According to MTT experiment,we drew the efficacy curve of gemcitabine and cisplatin successfully,and calculated that the ratio of the half-maximum inhibitory concentration(IC50)of gemcitabine and cisplatin were 0.4596mg/L and 0.2347mg/L,respectively.Therefore,according to the pharmacological principle,the species load ratio of gemcitabine and cisplatin in electrospinning sustained release system was about 2:1.Through many times of pre-experiments,the drug sustained-release materials with nanofibrous structure loaded with gemcitabine and cisplatin were successfully constructed by electrospinning technology.Under scanning electron microscope,we can observed that the fibers in the material were staggered into a network with uniform thickness,smooth surface,and no obvious beading defects,which is similar to the blank material.In terms of the quantitative diameter of nanofibers,the average diameters of fibers in control materials and sustained release materials were in the range of 0.8770±0.2299 and 0.3587±0.1264 um,respectively.The extraction process was repeated 10 times,the total mass of gemcitabine in all ultrapure was calculated as 4.188 ug,and the load drug rate of the materials was about 93.7%.The drug release curve of the sustained-release material was successfully drawn by high performance liquid chromatography(HPLC).According to the curve,it can be observed that the cumulative release of gemcitabine increased rapidly from 0 to 8 hours,The dissolution equilibrium of the material was reached when dissolved for 8 h,and the cumulative release of gemcitabine in solution did not increase significantly over time.The concentration of gemcitabine was about 0.295 mg/ml when reaching the dissolution equilibrium.Conclusion:By electrospinning technology,we successfully created the drug sustained release materials with nanofibrous structure,which not only have good characterization and diameter distribution,but also could load gemcitabine and cisplatin.This noval material has good drug loading rate,stable drug sustained-release ability and sustained release characteristics.Part 2To explore the local chemotherapy effect of the drug sustained-release materials with nanofibrous structure loaded with gemcitabine and cisplatin on the positive margin in solid tumorsObjective:To investigate the anti-tumor effect and the local chemotherapy effect of the drug sustained-release materials with nanofibrous structure loaded with gemcitabine and cisplatin on solid tumor with the positive margin in vitro.Methods:The mouse bladder cancer cells(MB49)and the breast cancer cells(4T1-luc)were used in the in vitro experiment and cultured in six-well plate,The two types of cells were randomly divided into four groups,respectively:Control group without treatment,negative control group(PLA group treated with a round slice of the drug-free electrospinning in medium),experimental group(PLA-drug group treated with a round slice of the drug sustained-release materials in medium),and positive control group(Drug group treated with 0.8 mg GEM and 0.4mg CDDP).The cells in four groups was collected in the 1st,2nd,and 3rd day,respectively,after treatment.The apoptosis rate was detected by flow cytometry,and the expression of apoptosis marker Caspase3 was detected by Western blot,in order to explore the anti-tumor effect of electrospinning drug sustained-release materials in vitro.To simulate positive surgical margins,we removed three quarters of the tumor volume in bladder cancer mouse model and mouse breast cancer mouse model with the operation.Then the mices were randomly divided into four groups with different treatments,which were the same as the above-mentioned cell groups:control group,PLA group,PLA-drug group,and drug group,in which drug group was injected intraperitoneally.After three weeks,the local chemotherapy and therapeutic effects of the drug sustained-release materials on positive margin of solid tumor was explored by animal fluorescence imaging,tumor volume monitoring and Tunel apoptosis fluorescence staining.Results:In vitro experiment of mouse breast cancer cell line 4T1-luc,on the 3rd day after treatment,the apoptotic cells in the PLA-drug group reached the same level as that in the Drug group,and the apoptosis rates in PLA-drug group and Drug group were 30.6%and 27.1%,respectively,which were significantly higher than those in the Control group and PL A group.During the three days in vitro experiment,with the increase of culture time,the expression of Caspase3 decreased continuously in the PLA-drug group,in the third day,the expression of Caspase3 in the PLA-drug group was lower compared with the Drug group(P=0.021).Similar results were also detected in bladder cancer cell lines in vitro.The apoptotic cells of the MB49 cells in the PLA-drug group and Drug group were increasing during the 3 days,and the apoptosis rates of the PLA-drug group and Drug group were 48.8%and 42.2%,respectively.In vivo experiment of mouse breast cancer model,fluorescence imaging of animals showed that in the two week after surgery,there were significant differences in the fluorescence intensity between PLA-drug group and PLA group(P=0.022),Drug group and Control group(P=0.035),PLA-drug group and Drug group(P=0.038),which suggested that that both drug sustained-release material group and intraperitoneal injection group had good therapeutic effect,and the therapeutic effect of s drug sustained-release material group was much better than that of intraperitoneal injection group.Similar results were obtained in the volume measurement of tumor model in three weeks after operation,there were significant differences in tumor volume between PLA-drug group and PLA group,Drug group and Control group(P<0.001 and P<0.021,respectively),but there was no significant difference between PLA-drug group and Drug group.Tunel apoptotic fluorescence staining of mouse tumor tissue sections showed that the ratio of apoptosis area in PLA-drug group was significantly larger than that in PLA group(P<0.001),and that in Drug group was significantly larger than that in Control group(P<0.001).In the mouse model of bladder cancer,the drug sustained-release materials showed similar effects.In three weeks after operation,there were significant statistical differences in tumor volume between PLA-drug group and PLA group,between Drug group and Control group,and between PLA-drug group and Drug group(P=0.012,P=0.008 and P=0.041).Conclusion:The study indicated the sustained-release materials with nanofibrous structure loaded with gemcitabine and cisplatin could effectively inhibit tumor growth,and also inhibit the growth of residual tumor with positive margin.It's local chemotherapy effect on the residual tumor was much better than that of direct intraperitoneal injection.Part 3To explore the influences of local administration of the drug sustained-release materials with nanofibrous structure on tumor immune microenvironment and the improvement of side effects of systemic chemotherapy.Objective:This study aimed to explore the influences of local administration of the drug sustained-release materials with nanofibrous structure on tumor immune microenvironment and the improvement of side effects of systemic chemotherapy.Methods:A mouse model with positive margin of breast cancer was established by removing three quarters of the tumor volume as mentioned above.The nude mice model were randomly divided into four groups and given different treatments,which were the same as the PART2.After the mice were treated for 3 weeks,blood samples were collected to detect liver and renal function and blood routine.Tumor,liver,kidney and blood samples of mice were collected,and the cumulative concentration of gemcitabine in tumor,liver,kidney and blood samples was determined by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).The collected tumor samples were fixed,embedded and sliced,and the distributions of bone marrow-derived suppressor cells(MDSCs)in tumor tissues of mice in each group were identified by double-labeled fluorescence staining co-localization technology,and the distribution of CD8+T cells and NKp46+NK cells in tumor tissues was detected by immunohistochemistry.Results:Among the various indexes of liver function in mice,the index of AST in PLA-drug group was significantly lower than that of Drug group(272.08±34.57 vs 186.76± 18.09,U/L;P=0.019),the index of ALT and AST in the mice of Control group were significantly higher than those of PLA group,which were associated with the presence of tumor liver metastasis in some mice in Control group,which was confirmed by previous animal imaging.Among the renal function indexes,the index BUN in PLA-drug group was significantly lower than that of Control group(5.52±7.238 vs 21.76±3.443,mg/dl;P=0.014).Among the blood routine indexes,the indexes of RBC and PLT in PLA-drug group were significantly higher than those of Drug group(5.53(5.175,5.73)VS 6.8(6.595,6.920),1012/L;P=0.031),(416(390,491.5)vs 751(735,849.5),109/L;P=0.019),and the RBC and PLT indexes in Control group were much similar to those in PLA group.ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)showed that the contents of gemcitabine in liver,kidney and serum in Drug group were significantly higher than those in PLA-drug group.Liver:(1509.97± 176.42 vs 813.48190.71,pg/g;P<0.001),kidney:(502.39±55.41 vs 235.52±34.93,pg/g;P<0.001),serum:(1729.06± 126.33 vs 1208.02±88.73,pmol/L;P<0.001).However,the content of gemcitabine in tumor samples of Drug group only account for 1/10 of that in PLA-drug group(832.93±79.14 vs 6993.01±337.08,pg/g;P<0.001).The double-labeled fluorescence staining showed that the distribution of MDSCs cells in PLA-drug group was significantly less than that in the other three groups,but there was no significant difference in the number of MDSCs cells between Drug group and Control group.Immunofluorescence histochemistry showed that the distribution of CD8+T cells in PLA-drug group was significantly higher than that in Drug group(P=0.013),and the distribution of NK cells in PLA-drug group was significantly higher than that in PLA group(P=0.032).Conclusion:Local administration of the drug sustained-release materials with nanofibrous structure can significantly improve the drug delivery efficiency,reduce the accumulation of drugs in normal organs and tissues,and reduce the damage of liver and kidney function and hematopoietic function caused by systemic chemotherapy.The local chemotherapy of this materials can also change the local immune microenvironment of tumor,destroy the growth environment of tumor cells,and enhance the killing effect of auto-immune system on tumor cells. |
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