双胍基偶联牛血清白蛋白载药纳米粒的制备及其抗肿瘤作用研究

发布时间：2018-06-29 14:42

本文选题：尼达尼布 + 双胍　；参考：《江苏大学》2017年硕士论文

【摘要】：双胍类是常用的治疗糖尿病药物之一,近年该类药物具有抗癌疗效常有报道,其结构中的双胍基被认为是一种具有潜在抗肿瘤作用的化学基团,同时该基团对磷酸根骨架的核酸分子具有高亲合性,且对磷脂双分子层结构的细胞膜具有较强穿透能力。尼达尼布(Nintedanib,NDNB)是用于特发性肺纤维化治疗的小分子化学药物,作为一种可抑制血管生成和组织纤维化的三重受体酪氨酸激酶抑制剂,大量临床前及临床研究已表明其具有较好的抗肿瘤作用。本课题先以双氰胺为底物合成了含双胍基的中间体,再采用混合酸酐法将其与牛血清白蛋白偶联,以其作为材料制备纳米粒运载药物NDNB,并对其体内外药动药效进行研究。课题旨在构建偶联改性的白蛋白纳米载体,增强NDNB抗肿瘤作用。论文分为以下几部分:第一部分综述本部分主要对双胍基的抗肿瘤作用的研究、偶联/修饰白蛋白载体的研究、NDNB的抗肿瘤作用研究进行介绍。了解双胍类药物及双胍基抗肿瘤的研究前沿,蛋白偶联/修饰方法及设计思路,NDNB的性质、药理作用及药动学性质等,为后续制剂研究进行理论准备。第二部分处方前研究本部分建立了紫外分光光度法作为体外药物含量检测的分析方法。通过紫外可见全波长扫描确定药物最大吸收波长为385nm,以此波长作为药物检测波长。配制相应不同浓度的药物溶液建立标准曲线,并对建立的含量测定方法进行方法学验证考察。实验表明所建立的体外药物含量测定方法合理可靠,符合方法学要求。对NDNB的释放介质中溶解度的考察结果表明NDNB在纯水中溶解度大,在磷酸盐溶液中的溶解度非常小,加入表面活性剂吐温80可增加其溶解度。第三部分双胍基偶联牛血清白蛋白的制备本部分先以双氰胺与对氨基苯甲酸为底物,反应合成具有潜在抗肿瘤活性的双胍基中间体对双胍基苯甲酸(DP),再采用混合酸酐法将其与牛血清白蛋白偶联(DP-BSA),采用薄层色谱法、红外光谱、核磁谱、质谱、紫外扫描、高效液相色谱法对合成中间体的结构及纯度进行表征,采用紫外及凝胶电泳对蛋白偶联产物DP-BSA进行确认表征,采用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和紫外扫描对DP-BSA的偶联度进行分析估算。经投料比、反应时间、反应pH环境单因素考察,确认最佳工艺处方,所得DP-BSA的偶联度为42.19。另外,还对DP-BSA溶解性等性质进行了初步考察。第四部分载药纳米载体的制备及体外释放本部分使用双胍基偶联白蛋白为载体材料制备双胍基偶联白蛋白纳米粒DP-BSANPs运载抗肿瘤模型药物NDNB,并通过使用激光粒径分析仪、透射电镜及紫外分光光度法对尼达尼布载药纳米粒(NDNB-DP-BSANPs)溶液的粒径分布、包封率、载药浓度进行检测表征。以粒径分布、形态、包封率、载药浓度为指标对处方及制备工艺进行单因素考察以选择孵化法最佳处方及工艺。结果显示,所制纳米粒平均粒径为47.18nm,多分散系数为0.15,平均载药浓度为1.586mg/mL,48h内含药量稳定。此外,以紫外分光光度法作为药物体外释放检测方法,通过透析法对于NDNB原料药溶液及载药纳米粒溶液在PBS(0.5%Tween80)溶液中的释放进行考察,体外药物释放结果表明载药纳米粒具有缓释作用。第五部分体内药物动力学考察本部分建立高效液相色谱法进行血药浓度检测,SD大鼠尾静脉给药进行体内药动学考察。方法学验证结果表明,所建立的方法专属性强、精密度高、回收率高、稳定性良好。体内药动学实验结果显示,NDNB在大鼠体内的药物动力学过程符合双室模型,给药后载药纳米粒组各时间点血药浓度均高于原料药组,原料药组半衰期为2.526h,载药纳米粒组半衰期4.588h;原料药组平均滞留时间为2.914h,明显低于载药纳米粒组为5.905h,载药纳米粒组相对生物利用度为162.96%,表明载药纳米粒可以增加药物的半衰期和生物利用度。第六部分体内抗肿瘤模型药效学研究及细胞实验本部分通过动物抗肿瘤模型给药实验及细胞实验对合成的偶联蛋白纳米载体及其NDNB载药纳米制剂抗肿瘤效果进行研究。抗肿瘤药效实验和细胞实验结果显示,双胍基偶联白蛋白载体对于肿瘤细胞有一定的抑制作用,载药纳米粒在两个实验中表现出较好的肿瘤生长抑制作用。
[Abstract]:Metformin is one of the commonly used drugs for the treatment of diabetes. In recent years, the antitumor effect of this kind of drugs is often reported. The metformin in its structure is considered as a chemical group with potential antitumor effect. At the same time, the group has high affinity to the nucleic acid molecule of the phosphate skeleton, and it has the cell membrane of the phospholipid bimolecular layer structure. Strong penetration. Neda Knibb (Nintedanib, NDNB) is a small molecular chemical used for the treatment of idiopathic pulmonary fibrosis. As a three receptor tyrosine kinase inhibitor that inhibits angiogenesis and tissue fibrosis, a large number of preclinical and clinical studies have shown that it has better anti-tumor effects. The intermediates containing bis guanidine were synthesized for the substrate, and then they were coupled with bovine serum albumin by the mixed anhydride method. The nanoscale carrier drug NDNB was prepared as a material, and the pharmacokinetics of the nanoparticles were studied. The aim of the study is to construct the modified albumin nanoscale and enhance the anti-tumor effect of NDNB. The paper is divided into the following parts Part 1: the first part is a summary of the research on the antitumor effect of metformin, the study of coupling / modified albumin carrier, the research on the anti-tumor effect of NDNB. The research frontier of the Antitumor of guanidine and metformin, the method of protein coupling / modification and the thinking, the properties of NDNB, the pharmacological action and the pharmacokinetic properties of the metformin In the second part, the second part of the study set up an analytical method for the determination of the drug content in vitro by UV spectrophotometry. The maximum absorption wavelength of the drug was determined by UV visible full wavelength scanning, and the wavelength was used as the detection wavelength of the drug. The standard curve of the solution was established and the method of determination was verified. The experiment showed that the method of determination of drug content in vitro was reasonable and reliable. The results of solubility in the release medium of NDNB showed that the solubility of NDNB in pure water was large and the solubility in phosphate solution was very good. Small, adding surface active agent Twain 80 can increase its solubility. Third the preparation of bis guanidine conjugated bovine serum albumin (BSA) in this part first reaction with dicyandiamide and p-aminophenic acid as substrate, the synthesis of dianidine intermediate with potential antitumor activity against guanidine benzoic acid (DP), and then mixed anhydride with bovine blood white eggs DP-BSA, the structure and purity of the synthetic intermediates were characterized by TLC, IR, NMR, MS, UV, and high performance liquid chromatography. The protein coupling product DP-BSA was characterized by UV and gel electrophoresis, and the base assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was used. The coupling degree of DP-BSA was analyzed and estimated by UV scanning. The optimum process was confirmed by the ratio of feeding, reaction time and reaction of pH environment. The coupling degree of DP-BSA was 42.19., and the solubility of DP-BSA was preliminarily investigated. The preparation of the fourth part of drug carrying nanoscale and the release of this part in vitro were used in two parts. Guanidine coupling albumin was used as carrier material to prepare antitumor model drug NDNB, which was loaded with guanidine coupling albumin nanoparticles DP-BSANPs. The particle size distribution, encapsulation efficiency and drug concentration of Neda Knibb loaded nanoparticles (NDNB-DP-BSANPs) solution were detected by laser particle size analyzer, transmission electron microscope and ultraviolet spectrophotometry. The optimum formulation and process of the formulation and preparation process were selected by the particle size distribution, morphology, encapsulation efficiency and drug loading. The results showed that the average particle size of the nanoparticles was 47.18nm, the polydispersity coefficient was 0.15, the average drug concentration was 1.586mg/ mL, and the content of the 48h was stable. In addition, UV spectrophotometry was used. As a method of drug release detection in vitro, the release of NDNB drug solution and drug loaded nanoparticles solution in PBS (0.5%Tween80) solution was investigated by dialysis. The drug release in vitro showed that the drug loaded nanoparticles had sustained release effect. The fifth part of the pharmacokinetic study in vivo was established by high performance liquid chromatography The pharmacokinetic study of the tail vein of SD rats was conducted in vivo. The results showed that the established method was highly exclusive, high precision, high recovery and good stability. The pharmacokinetic experiment in vivo showed that the pharmacokinetics of NDNB in rats was in accordance with the double chamber model, and the drug loaded nanoparticles were given each time after the drug was given. The concentration of blood drug was higher than that of the drug group, the half-life of the drug group was 2.526h, the half life of the drug loaded nanoparticles group was 4.588h, the average retention time of the drug drug group was 2.914h, which was significantly lower than the drug loaded nanoparticles group was 5.905h, and the relative bioavailability of the drug loaded nanoparticles group was 162.96%, indicating that the drug loaded nanoparticles could increase the half-life and biological benefits of the drug loaded nanoparticles. Sixth part of the antitumor model in vivo and cell experiment in this part, the anti tumor effect of the synthesized coupling protein nanoscale carrier and its NDNB drug loaded nanoscale preparation was studied by the animal antitumor model administration and cell experiment. The antitumor effect experiment and the cell experiment result showed that the double guanidine coupling albumin was found. The carrier has a certain inhibitory effect on tumor cells. Drug loaded nanoparticles show a good inhibitory effect on tumor growth in two experiments.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;TQ460.1

【参考文献】