聚甲基丙烯酸甲酯骨水泥作为化疗药物复合载体的研究

发布时间：2018-03-25 07:56

本文选题：药物复合聚甲基丙烯酸甲酯骨水泥　切入点：甲氨蝶呤　出处：《武汉大学》2016年博士论文

【摘要】：目的：筛选出热稳定性佳且可被复合入聚甲基丙烯酸甲酯(PMMA)骨水泥的化疗药；观察甲氨蝶呤(MTX)和盐酸表柔比星(EPI)分别或者联合与PMMA骨水泥以不同质量比例混合制成的复合化疗药PMMA骨水泥的机械强度和显微结构,探索MTX和EPI可复合入PMMA骨水泥的较大添加量；观察MTX和EPI复合PMMA骨水泥的药物体外模拟释放特性和释放药物活性,并对比释放前后骨水泥机械强度和显微结构的变化；为制作可临床应用的MTX和EPI复合PMMA骨水泥提供理论依据。方法：1、将不同浓度的实验组MTX (10、20、40、80、160μg/ml)和EPI(1.25、2.5、5、10、20μg/ml)溶液在75℃的条件下热处理10min之后,同时对照组在室温下静置10min之后,分别将上述药品溶液加入MG63和U2OS两种细胞中培养24、48h后使用CCK8试剂盒进行细胞毒性检测。2、分别将MTX与PMMA骨水泥以1%、2%、4%的质量比复合；EPI与PMMA骨水泥以0.1%、0.2%、0.4%的质量比复合； MTX联合EPI两种药物与PMMA骨水泥以1%MTX+O.1%EPI、2%MTX+0.2%EPI、4%MTX+0.4%EPI的质量比复合,以及未复合药品的骨水泥分别制成骨水泥标准试件分别检测计算其机械性能(抗压强度、抗弯强度和抗弯模量)及观察电镜下微结构的改变。3、分别将MTX与PMMA骨水泥以2%的质量比复合、EPI与PMMA骨水泥以0.1%的质量比复合的PMMA骨水泥制成圆柱形骨水泥测试标准试件各5个,称量并记录每一个标准试件的质量。将每个标准试件放入10ml的离心管中,加入10ml的37℃生理盐水,每隔24h换液,分别将第1、2、3、5、7、10、15、20、30天时的释放洗脱液置入-20℃冰箱中保存。使用高效液相色谱仪对置换出来的液体进行定性及定量测试。4、将定量定性测试后的药物释放洗脱液加入143b细胞系中培养24h,同对照组作对比,采用CCK8试剂盒检测其药物活性。5、分别将MTX与PMMA骨水泥以2%的质量比复合、EPI与PMMA骨水泥以0.1%的质量比复合的PMMA骨水泥制成圆柱体和长方体骨水泥测试标准试件各5个。将每个标准试件放入10ml的离心管中,加入10ml的37℃生理盐水,并且每隔24h换液,持续5个月后检测并计算其机械性能(抗压强度、抗弯强度和抗弯模量)及电镜下微结构的改变。结果：1、不同浓度的两种化疗药在进行75℃处理后,分别于24h和48h采用CCK8试剂盒测试,方差分析其结果显示各组MTX和EPI热处理后细胞毒性实验结果同对照组差异无统计学意义(P0.05)。表明MTX和EPI在75℃下处理10分钟后其活性均无明显降低。2、MTX和EPI分别或联合与PMMA骨水泥以质量比不高于4%和0.4%复合后,复合骨水泥各项机械性能指标均高于国际参考值(抗压强度70MPa,抗弯强度50MPa,≥1800MPa)。以抗压强度为因变量,采用回归分析发现,MTX和EPI与PMMA骨水泥复合质量比对抗压强度无显著影响(P=0.120,P=0.951)。以抗弯强度为因变量,采用回归分析发现MTX与PMMA骨水泥复合质量比对抗弯强度无显著影响(P=0.912)；EPI与PMMA复合质量比对抗弯强度有显著影响(P=0.029,13=2315.305),EPI在一定程度上可加强PMMA骨水泥的抗弯强度,以抗弯模量为因变量,采用回归分析发现,MTX和EPI与PMMA骨水泥复合质量比对抗压强度及抗弯模量无显著影响(P=0.947,P=0.556)。电镜观察下未复合化疗药的PMMA骨水泥表面或断面上分布着大量不规则孔隙；复合入MTX后的PMMA骨水泥表面或断面上的不规则孔隙变浅变少;复合入EPI后的骨水泥表面或断面上的不规则孔隙明显变浅变少;随着药品量的增加,不规则空隙会进一步变浅变少。3、MTX和EPI均可以从骨水泥中释放洗脱出来,其中MTX在第一天释放峰值出现之后便急剧下降后趋于平缓稳定,EPI则始终较为稳定,未有较高峰值出现。4、MTX和EPI在第三十天的释放洗脱液加入143b培养细胞中培养24h,t检验分析其结果显示实验组和对照组细胞存活率差异有显著统计学意义。说明骨水泥释放药物具有长期抗肿瘤作用。5、MTX与PMMA骨水泥以2%的质量比复合、EPI与PMMA骨水泥以0.1%的质量比复合的PMMA骨水泥在药物释放前后,复合骨水泥各项机械强度性能指标均高于国际参考值(抗压强度70MPa,抗弯强度50MPa,≥1800MPa)。同对照组相比较药物复合骨水泥在药物释放洗脱后表面孔隙中的药物颗粒明显减少。结论：甲氨蝶呤(MTX)和盐酸表柔比星(EPI)在PMMA骨水泥聚合时产生的长时高温环境下生物活性不会明显降低,可以被复合入PMMA骨水泥来制作化疗药复合骨水泥；分别或者联合,MTX与PMMA骨水泥以不超过4%的质量比复合,EPI与PMMA骨水泥以不超过0.4%的质量比复合,不会对PMMA骨水泥的机械强度产生不利影响；骨水泥中的MTX和EPI可以长期稳定地从骨水泥中释放出来并具备抗肿瘤效果；PMMA骨水泥可作为药品的良好载体。
[Abstract]:Objective: to screen a better thermal stability and can be mixed into polymethylmethacrylate (PMMA) bone cement chemotherapy; observation of methotrexate (MTX) and epirubicin hydrochloride (EPI) mechanical strength and microstructure of bone cement composite chemotherapy combined with PMMA or PMMA respectively with different ratio of bone cement mixed, exploration a large amount of MTX and EPI can be combined into PMMA bone cement; observation of MTX and EPI composite PMMA bone cement in vitro drug release characteristics and release of drug activity, and to compare the changes of bone cement mechanical strength and microstructure before and after release; for the production can provide a theoretical basis for the clinical application of MTX and EPI composite PMMA bone cement. Methods: 1, the experimental groups with different concentrations of MTX (10,20,40,80160 g/ml) and EPI (1.25,2.5,5,10,20 g/ml) solution after heat treatment 10min the temperature of 75 DEG C, and the control group at room temperature static The 10min, respectively, the drug solution was added to two kinds of MG63 and U2OS in cultured cells after 24,48h using CCK8 kit to detect the cytotoxic.2, respectively MTX and PMMA bone cement with 1%, 2%, 4%, the mass ratio of EPI and PMMA composite; bone cement with 0.1%, 0.2%, 0.4% mass ratio of the composite; MTX combined with EPI two kinds of drugs and PMMA bone cement with 1%MTX+O.1%EPI, 2%MTX+0.2%EPI, the mass ratio of 4%MTX+0.4%EPI composite bone cement composite, and no drugs were made from bone cement standard specimens were measured to calculate the mechanical properties (compressive strength, flexural strength and flexural modulus of.3 micro structure change) and observed under electron microscope, respectively. The MTX and PMMA bone cement with 2% mass ratio of EPI and PMMA composite bone cement with 0.1% of the mass ratio of PMMA bone cement composite cylindrical bone cement test standard test piece 5, weighing and recording every standard specimen quality. Each standard specimen in the 10ml centrifugal tube, 37 DEG C with saline 10ml, every 24h in liquid, respectively, the first 1,2,3,5,7,10,15,20,30 days of the release of elution preservation solution into the -20 C refrigerator. Using HPLC qualitative and quantitative test of.4 replacement out of the liquid, the qualitative and quantitative test of drugs the release of the eluent culture 24h joined the 143B cell line, compared with the control group, using CCK8 kit to detect the activity of drugs.5, MTX and PMMA respectively, with 2% of the mass ratio of bone cement composite, EPI and PMMA of bone cement with 0.1% of the mass ratio of PMMA bone cement composite made of cylindrical and rectangular bone cement standard test specimen of the 5. Each standard specimen in the 10ml centrifugal tube, 37 DEG C with saline 10ml, and every 24h was changed, continued 5 months after the test and calculate the mechanical properties (compressive strength, flexural strength And the flexural modulus) and change the micro structure under electron microscope. Results: 1. Two kinds of chemotherapeutic drugs at different concentrations of 75 DEG C after treatment, using CCK8 kit to test 24h and 48h respectively, the variance analysis results of each group of MTX and EPI after heat treatment, cell toxicity test results showed no difference with the control group meaning (P0.05). The results indicated that MTX and EPI at 75 DEG C for 10 minutes after the activity did not significantly decrease.2, MTX and EPI respectively or combined with PMMA bone cement in the ratio is not higher than 4% and 0.4% compound, the mechanical properties of bone cement composite index was higher than the international reference value (anti compressive strength 70MPa the bending strength is 50MPa, or 1800MPa). The dependent variable by compressive strength is found by regression analysis, no significant effect of MTX and EPI and PMMA composite bone cement ratio on compressive strength (P=0.120, P=0.951). The dependent variable bending strength was found, MTX and PMMA bone by regression analysis Composite cement mass ratio on flexural strength had no significant effect (P=0.912); EPI and PMMA composite quality have significant effect on the bending strength ratio (P=0.029,13=2315.305), EPI can enhance the flexural strength of PMMA bone cement in a certain extent, with the dependent variable bending modulus is found by regression analysis, no significant effect on MTX and EPI with PMMA bone cement ratio on the compressive strength and flexural modulus (P=0.947, P=0.556). Electron microscope is not combined with chemotherapy of PMMA bone cement surface or on the cross section of the distribution of a large number of irregular pores; composite bone cement into the PMMA section on the surface or irregular pores after MTX shallower and less into the composite; the cement surface or section of irregular pore after EPI significantly shallower and less; with the increase of drug consumption, irregular gap will further shallower and less.3, MTX and EPI were eluted can be released from bone cement, in which MTX After the first day of release peak decreased rapidly after gently stable, EPI remained stable, no high peak of.4, MTX and EPI on the thirtieth day of the release of the eluent into 143B in cultured 24h cells, t analysis showed statistically significant for the experimental group and the control group difference in survival rate. Bone cement release drugs have long-term antitumor effects of.5, MTX and PMMA to 2% mass ratio of bone cement composite, EPI and PMMA to 0.1% mass ratio of bone cement composite PMMA bone cement in drug release and the mechanical strength of the composite bone cement performance index are higher than the international reference value (anti compressive strength 70MPa the bending strength is 50MPa, or 1800MPa). Compared with the control group of drug particles in the surface pore drug release after elution of the drug composite bone cement was significantly reduced. Conclusion: Methotrexate (MTX) and epirubicin acid salt Star (EPI) in the PMMA bone cement polymerization long without biological activity under high temperature significantly reduced, can be incorporated into PMMA bone cement to make chemotherapy combined with bone cement; separately or in combination, MTX and PMMA of bone cement to no more than 4% of the mass ratio of EPI and PMMA composite bone cement with no more than 0.4% the mass ratio of composite, does not adversely affect the mechanical strength of PMMA bone cement; bone cement in MTX and EPI can be stably for a long period of time and released from the bone cement have anti-tumor effect; PMMA bone cement can be used as a good drug carrier.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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