肝靶向羧甲基壳聚糖衍生物自组装纳米粒给药系统的研究

发布时间：2018-03-05 06:47

本文选题：肝靶向　切入点：羧甲基壳聚糖衍生物　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：自组装聚合物纳米粒是由两亲性聚合物在水中通过疏水作用、静电作用、氢键作用、金属配位作用等驱动力,自组装(或自聚集)形成具有亲水性外壳和疏水性内核的壳-核型纳米微粒。自组装聚合物纳米粒具有较高的热力学稳定性,内核可以作为疏水性药物、基因、多肽和蛋白质的贮库,亲水性外壳具有延长体内循环时间的作用,并且可以通过化学修饰达到主动靶向、微环境响应(pH敏感、温度敏感、磁靶向)、逃避单核细胞的吞噬、提高生物膜转运等目的,因此自组装聚合物纳米粒在药物递送方面具有重大潜力,受到越来越多科研工作者的关注。本文以水溶性O-羧甲基壳聚糖为骨架材料,对其进行疏水性和靶向性修饰,用于构造自组装聚合物纳米粒,作为抗肿瘤药物的载体。首先,利用EDC催化的酰胺化反应先后将具有疏水性烷基链的硬脂酸和具有肝靶向功能的乳糖酸接枝到O-羧甲基壳聚糖骨架上。利用红外光谱法、核磁共振法以及元素分析法确证聚合物的结构。采用超声法制备自组装纳米粒并对其粒径、表面电位进行表征。由O-羧甲基壳聚糖接枝硬脂酸聚合物(OS)制备的纳米粒粒径在168～216nm范围内,随着硬脂酸取代度的增多,粒径先减小后增大。选取硬脂酸取代度为9.8%的OS聚合物进一步修饰半乳糖,得半乳糖化O-羧甲基壳聚糖接枝硬脂酸聚合物(Gal-OS),半乳糖取代度为13.1%。以此材料制备肝靶向自组装聚合物纳米粒,粒径约为160nm。采用芘荧光探针法测量聚合物的临界聚集浓度。随着硬脂酸取代度的增加,OS聚合物CAC逐渐减小,而Gal-OS聚合物比具有相同硬脂酸取代度的未经乳糖酸修饰的OS聚合物的CAC略大。溶血实验结果显示,OS聚合物和Gal-OS聚合物的溶血率低于5%,说明材料血液相容性较好,符合静脉注射的要求。空白材料的细胞毒性实验表明,所制备的两种材料对细胞没有明显的细胞毒性,可以应用于生物医学领域。将多柔比星作为治疗药物,制备Gal-OS/DOX自组装纳米粒。纳米粒载药量为13.4%,包封率为77.4%,粒径为181.9nm。透射电镜观察纳米粒形态,为类球形实体,表面光滑、纳米粒间不粘连。采用透析法考察Gal-OS/DOX和OS/DOX自组装纳米粒在三种不同pH值的释放介质中的释放行为。结果表明,Gal-OS/DOX和OS/DOX纳米粒体外释放具有缓释性,并且在较低pH的条件下释药更快,即具有pH敏感性。本研究建立了荧光分光光度法测定多柔比星在血浆样品和组织样品中含量的分析方法。大鼠体内药动学研究结果显示,Gal-OS/DOX和OS/DOX纳米粒均能显著延长多柔比星的体内循环时间,降低清除速率。其药-时曲线下面积分别为多柔比星溶液组的29倍和31倍。小鼠组织分布实验结果表明,Gal-OS/DOX和OS/DOX纳米粒均能增加DOX在肝和脾的分布。其中,OS/DOX纳米粒组在肝脏的AUC是溶液组的7.3倍,而Gal-OS/DOX纳米粒组在肝脏的AUC是溶液组的9.8倍,表明Gal-OS/DOX纳米粒比OS/DOX纳米粒更能增加药物在肝脏的积蓄。除此之外,两种纳米粒在心脏、肾脏以及肺的AUC比溶液组均要小,表明载药纳米粒均能降低药物对心、肾以及肺的毒性,尤其是心脏毒性的降低,对多柔比星临床应用具有重大意义。靶向性评价结果说明,载药纳米粒对肝脏的趋向性大于多柔比星溶液组,且Gal-OS/DOX对肝脏的趋向性大于OS/DOX。本文首次合成并报道了由乳糖酸、硬脂酸、O-羧甲基壳聚糖构筑的新型肝靶向材料半乳糖化O-羧甲基壳聚糖接枝硬脂酸聚合物,并以此作为抗肿瘤药物多柔比星的载体,制备肝靶向羧甲基壳聚糖衍生物自组装纳米粒制剂。本研究成果将为多柔比星的临床应用以及进一步探讨肝靶向给药系统提供参考和新的选择。
[Abstract]:The self-assembly of polymer nanoparticles is composed of two amphiphilic polymer in water by hydrophobic interaction, electrostatic interaction, hydrogen bonding, metal complexation, driving force, self assembly (or self aggregation) formed with hydrophilic shell and hydrophobic core shell nanoparticles. Karyotype thermodynamic stability of self-assembled polymer nanoparticles with high the kernel, can be used as hydrophobic drugs, gene, storage of peptides and proteins, the hydrophilic shell can prolong the circulation time in vivo, and can be reached by chemical modification of active targeting, response of micro environment (pH sensitive, temperature sensitive, magnetic targeting), phagocytosis of mononuclear cells escape, improve the biological membrane etc, so it has a great potential of self-assembled polymeric nanoparticles in drug delivery, more and more researchers' attention.
In this paper, the water soluble O- carboxymethyl chitosan as matrix material, the hydrophobic and targeted modification, for the construction of self assembled polymer nanoparticles as carrier of anti-tumor drugs. Firstly, stearic acid by amidation reaction catalyzed by EDC has a hydrophobic alkyl chain and lactose acid with liver graft targeting function to O- carboxymethyl chitosan skeleton. By infrared spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis confirmed the structure of the polymer. The ultrasonic preparation of self-assembled nanoparticles and their particle size and surface potential were characterized by O- carboxymethyl chitosan grafted stearic acid polymer (OS) prepared the particle size in the range of 168 ~ 216nm, with the degree of substitution of stearic acid increased, the particle size decreased and then increased. The degree of substitution of OS for selecting stearic acid polymer 9.8% galactose was further modified, galactosed O- carboxymethyl chitosan base Sugar grafted stearic acid polymer (Gal-OS), galactose substitution degree of 13.1%. taking the material preparation of liver targeted self-assembled polymeric nanoparticles, the particle size is about critical aggregation concentration of 160nm. using pyrene fluorescence probe method for the measurement of polymer. With the increase of degree of substitution of stearic acid, OS polymer CAC and Gal-OS gradually decreased, compared with the same polymer the degree of substitution of stearic acid without OS polymer lactose acid modified CAC slightly. The hemolysis experiment results show that OS polymer and polymer Gal-OS hemolytic rate of less than 5%, indicating the material has better blood compatibility, with intravenous injection requirements. Showed that the cytotoxicity of blank material, two kinds of materials prepared by no cell obvious toxicity to cells, can be used in the biomedical field. Doxorubicin as a treatment drug, preparation of Gal-OS/DOX nanoparticles self-assembled nanoparticles. The drug loading was 13.4%. The entrapment efficiency was 77.4%, grain The size of 181.9nm. transmission electron microscope to observe the morphology of nanoparticles were spherical, solid, smooth surface, no adhesion between the nanoparticles. The dialysis method was used to study the Gal-OS/DOX and OS/DOX self-assembled nanoparticles in three kinds of release medium of different pH value in the release behavior. The results showed that Gal-OS/DOX and OS/DOX nanoparticles in vitro release is sustained, and at low pH under the condition of drug release faster, which is the pH sensitivity.
This study established a method to measure than the stars in the plasma samples and tissue samples in soft fluorescence spectrophotometry. Pharmacokinetics in rats showed that Gal-OS/DOX and OS/DOX nanoparticles can significantly prolong the circulation time and more flexible than the stars, reduce the clearance rate. AUCs were doxorubicin solution were 29 times and 31 times. The tissue distribution in mice. The experimental results show that Gal-OS/DOX and OS/DOX nanoparticles can increase the distribution of DOX in the liver and spleen. Among them, OS/DOX nanoparticles group in the liver of the AUC is 7.3 times solution group, Gal-OS/DOX nanoparticles group in the liver of the AUC is 9.8 times solution group, showed that Gal-OS/DOX nanoparticles can increase the drug savings in the liver more than OS/DOX nanoparticles. In addition, two kinds of nanoparticles in the heart, kidney and lung of AUC group was smaller than that of solution, showed that the drug loaded nanoparticles can reduce the drug The toxicity of heart, kidney and lung, especially the decrease of cardiac toxicity, is of great significance for the clinical application of doxorubicin. The results of targeted evaluation indicate that the tendency of drug loaded nanoparticles to liver is greater than that of doxorubicin solution group, and the tendency of Gal-OS/DOX to liver is greater than OS/DOX..
This paper was first synthesized and reported by lactose acid, stearic acid, O- carboxymethyl chitosan to construct new liver targeting material galactosed O- carboxymethyl chitosan grafted stearic acid polymer, and as anticancer drug doxorubicin carrier, preparation of liver targeted carboxymethyl chitosan derivatives self-assembled nanoparticles. The results of this study will be to doxorubicin in clinical application and further study of liver target provide reference and new options to the drug delivery system.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R94

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