多功能聚合物胶束载药体系的构建及其抗肿瘤研究

发布时间：2018-03-25 21:14

本文选题：肝癌靶向　切入点：pH敏感　出处：《滨州医学院》2015年硕士论文

【摘要】：本文旨在设计、合成功能高分子材料,构建多功能主动靶向抗肿瘤药物纳米递药系统。(1)利用肝癌细胞高表达特异性去唾液酸糖蛋白的特点,合成了含有其配体吡喃半乳糖的两亲性嵌段聚合物MPEG-b-PMaIPG,利用其自组装行为构建了一种肝癌靶向、pH敏感的多功能纳米载药体系。(2)利用恶性肿瘤细胞较正常细胞过度表达葡萄糖转运蛋白1(GLUT 1)的特点,将其配体N-乙酰-D-氨基葡萄糖(NAG)共价键合到苯乙烯-马来酸酐嵌段共聚物上,制备了主动靶向性聚合物NAG-P(St-alt-MA)58-b-PSt130纳米药物载体。第一部分利用原子转移自由基聚合(ATRP)技术,制备了一种分子量分布窄(Mw/Mn=1.21)的含糖嵌段共聚物。用核磁共振氢谱(1H NMR)及凝胶渗透色谱(GPC)对其结构进行了表征,确定嵌段共聚物分子式为MPEG42-b-PMaIPG20。利用荧光光谱、纳米粒径Zeta电位分析仪(DLS)、投射电子显微镜(TEM)等对其自组装行为进行了表征,实验测得该共聚物的临界胶束浓度为0.005 mg/mL;纳米粒径约为100±4.43 nm, Zeta电位为-32.8±0.23 mV。并以DOX为模型药物,构建了纳米载药体系,载药量为24.77±2.68%,包封率为66.12±9.44%。透射电子显微镜观察载药前后粒径分别约50 nm、60 nm球形纳米粒子。利用透析法考察了体外药物释放,72 h后不同pH环境下药物释放率分别为：pH 5.0时DOX释放率达85%,而在pH 6.5、pH 7.4环境下DOX的释放分别为65%、37%,表明该纳米载药纳米体系具有明显的pH敏感特性。红细胞溶血实验表明,浓度1.0 mg/mL时,MPEG42-b-PMaIPG2o纳米粒子的溶血率均小于5%,符合生物医用材料对溶血的要求。MTT法检测MPEG42-b-PMaIPG20纳米粒子对小鼠成纤维细胞(L929)及肝癌细胞(HepG2)在检测范围内细胞存活率均大于95%,均未表现出细胞毒性。利用激光共聚焦(CLSM)和流式细胞术(FCM)对载药纳米粒子DOX-NPs的摄取做了定性、定量测定,结果表明相同实验条件下,HepG2细胞对DOX-NPs的摄取率均高于A549细胞对DOX-NPs的摄取。以HepG2和A549细胞为细胞模型,考察了DOX-NPs和游离阿霉素(DOX)对细胞的生长抑制效果,结果显示两种测试样品对细胞的生长抑制均随药物浓度的增大而提高,DOX-NPs在实验浓度范围内对HepG2细胞产生较强的抑制效果,而对A549细胞的抑制作用则相对较小。第二部分制备一种新型的主动靶向聚合物纳米载药体系。通过FTIR和1HNMR对NAG接枝嵌段聚合物NAG-P(St-alt-MA)58-b-PSt130进行了结构表征。芘荧光探针荧光光度法测得该两亲性嵌段共聚物的临界胶束浓度为0.028 mg/mL。动态光散射测得粒径为56.27±0.43 nm, PDI为0.099,pH 7.4时纳米粒子表面Zeta电位为-41.46±0.99 mV。负载脂溶性药物DOX后,载DOX纳米粒子粒径为64.21 ±0.96 nm, PDI为0.10,载药量为25.83±1.09%,包封率为69.69±3.98%。透射电子显微镜观察载药前后纳米粒子均为球形,粒径大小分布均匀,分别为50 nm,60 nm。体外药物释放证明载药体系具有较好的缓释功能。体外细胞毒性试验证明该纳米载体在检测浓度范围内对ATⅡ细胞的生存率均80%,表现为低毒。以MCF-7细胞和HT29细胞作为模型细胞,利用激光共聚焦(CLSM)和流式细胞术(FCM)对载药粒子NAG-NPs-DOX和NPs-DOX的摄取进行了定性、定量分析,对比实验表明,相同实验条件下两种细胞对NAG-NPs-DOX的摄取均多于对NPs-DOX的摄取。同时考察了NAG-NPs-DOX、NPs-DOX对两种模型细胞的生长抑制效果,结果显示相同条件下NAG-NPs-DOX载药体系对肿瘤细胞的抑制率均强于NPs-DOX对细胞的抑制率。研究结果初步证实,含葡萄糖转运蛋白1(GLUT1)配体N-酰-D-氨基葡萄糖分子的NAG-P(St-alt-MA)58-b-PSt130纳米载体有利于肿瘤细胞吞噬,具有肿瘤细胞靶向性。本论文研究了两种基于受体-配体介导的肿瘤细胞靶向技术,通过体外实验评价了这两种载体材料的生物安全性和靶向性。实验结果表明,本文所研究的自组装聚合物纳米粒子有望成为集缓控释、药物靶向于一体的抗肿瘤药物载药系统。
[Abstract]:This paper aims to design and synthesis of functional polymer materials, construction of multifunctional active targeting drug delivery system to anticancer drugs. The use of nano (1) liver cancer cells with high expression of specific asialoglycoprotein, synthesized the ligand containing galactopyranose two amphiphilic block polymer MPEG-b-PMaIPG, the self-assembly behavior was constructed a target for liver cancer, multifunctional nano pH sensitive drug carrier system. (2) compared with the normal expression of glucose transporter 1 by malignant cells (GLUT 1) characteristics of the ligand N- acetyl -D- glucosamine (NAG) covalently bonded to the styrene maleic anhydride copolymer, active targeted polymer to prepare NAG-P (St-alt-MA) 58-b-PSt130 nano drug carrier. The first part using atom transfer radical polymerization (ATRP) technology, the preparation of a narrow molecular weight distribution (Mw/Mn= 1.21) containing sugar block copolymer. Using nuclear magnetic resonance spectroscopy (1H NMR) and gel permeation chromatography (GPC) were used to characterize its structure, determine the copolymer molecular formula MPEG42-b-PMaIPG20. by fluorescence spectroscopy and nano particle size Zeta potential analyzer (DLS), transmission electron microscopy (TEM) and the self assembling behaviors were characterized and measured the critical micelle concentration of the copolymer was 0.005 mg/mL; the particle size is about 100 + 4.43 nm, Zeta potential is -32.8 + 0.23 mV. and DOX as the model drug and construct nanoparticle drug delivery system, drug loading was 24.77 + 2.68%, the encapsulation rate of 66.12 + 9.44%. transmission electron microscope before and after drug particle diameters about 50 nm, 60 nm spherical nanoparticles were investigated. The in vitro drug release by dialysis, after 72 h of different pH under the environment of drug release rates were: pH 5 DOX release rate reached 85%, while in the pH 6.5 release of DOX under the environment of pH 7.4 were 65%, 37%, show PH has the obvious characteristics of the sensitive drug loaded nano nano system. That red blood cell hemolysis test, the concentration of 1 mg/mL, the hemolysis of MPEG42-b-PMaIPG2o nanoparticles were less than 5%, requirements for the hemolysis of biomedical materials.MTT detection of MPEG42-b-PMaIPG20 nanoparticles on small rat fibroblasts with (L929) and hepatocellular carcinoma (HepG2) in the detection range within the cell survival rate was more than 95%, showed no cytotoxicity. By confocal laser scanning microscope (CLSM) and flow cytometry (FCM) on the uptake of DOX-NPs nanoparticles to do a qualitative, quantitative analysis results show that under the same experimental conditions, the DOX-NPs uptake rate of HepG2 cells was higher than that of DOX-NPs uptake with HepG2 and A549 cells. A549 cells, the effects of DOX-NPs and free adriamycin (DOX) inhibitory effect on cell growth, the results showed that two kinds of test samples on cell growth inhibition Was increased with the increasing of drug concentration, DOX-NPs has strong inhibitory effect on HepG2 cells in the experimental concentration range, and the inhibitory effect on A549 cells is relatively small. The second part for the preparation of a novel active targeting polymer nano drug carrier system. Through the FTIR and 1HNMR of NAG NAG-P (graft copolymer St-alt-MA) 58-b-PSt130 were characterized. The pyrene fluorescence probe fluorescence spectrophotometry measured the two amphiphilic block copolymers of critical micelle concentration of 0.028 mg/mL. measured by dynamic light scattering particle size of 56.27 + 0.43 nm, PDI 0.099, pH 7.4 Zeta nano particle surface potential of -41.46 + 0.99 mV. fat soluble load drug DOX, DOX nanoparticle size was 64.21 + 0.96 nm, PDI was 0.10, the drug loading was 25.83 + 1.09%, the encapsulation rate of 69.69 + 3.98%. transmission electron microscope before and after drug loaded nanoparticles were spherical, the particle size of points Uniform, were 50 nm, 60 nm. demonstrated the in vitro drug release drug carrier system with slow-release function better. In vitro cytotoxicity test showed that the nanoparticles in the detection range of concentration of AT II cell survival rate were 80%, showed low toxicity. MCF-7 cells and HT29 cells as a model cell, using laser scanning focus (CLSM) and flow cytometry (FCM) quantitative analysis of drug loaded particles NAG-NPs-DOX and NPs-DOX uptake were qualitative, comparative experiments show that the two species under the same experimental conditions the cellular uptake of NAG-NPs-DOX were more than the uptake of NPs-DOX. At the same time, the effects of NAG-NPs-DOX, the inhibitory effect of NPs-DOX on the two cell model the growth results show that under the same conditions NAG-NPs-DOX drug carrier system inhibition rate of tumor cells was stronger than NPs-DOX inhibitory rate of cells. The results confirmed that the content of glucose transporter 1 (GLUT1) ligand N- acyl -D Glucosamine molecules NAG-P (St-alt-MA) 58-b-PSt130 nanoparticles to tumor cell phagocytosis, with tumor cell targeting. This paper studied two kinds of tumor cells based on the target receptor ligand mediated to technology, in vitro evaluation of these two kinds of carrier materials of biological safety and targeting experiments. The results show that the self-assembly of polymer nanoparticles is expected to become the set of controlled-release drugs, targeted anticancer drug delivery system in one.

【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943;R96

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