pHGSH响应羧甲基壳聚糖水凝胶及纳米凝胶的研究

I
万方数据
均粒径在 160nm 左右。 动态光结果显示纳米凝胶在 pH7.4 缓冲液中放置一个月前后的粒径和分散 系数均未发生较大变化，表明制备的纳米凝胶具有较好的稳定性，且在较低浓 度（5μM）谷胱甘肽缓冲液中，其平均粒径也未发送较大变化，而在含含 5mM 和 20mM 缓冲液中的平均粒径显著增大，显示出谷胱甘肽响应性。纳米凝胶的 粒径随着 pH 值变化而变化，表现出良好的 pH 敏感性，且其敏感程度与巯基化 羧甲基壳聚糖中巯基含量有关。 利用两亲性大分子在水溶液中自组装成纳米粒子，经原位交联形成核壳型 纳米凝胶，该制备方法简单，且无需使用交联剂。制备的纳米凝胶具有 pH 和谷 胱甘肽双重响应性，考虑到肿瘤细胞与正常细胞内环境的差异，可将该纳米凝 胶作为肿瘤细胞内的药物控释载体。纳米凝胶的表面有大量的羧基，可对进行 化学修饰链接配体，起到靶向传递效果。 （4）以甲氨蝶呤为药物模型，研究了pH/GSH响应羧甲基壳聚糖载药纳米凝 胶在模拟人体血液、正常细胞内和肿瘤细胞内介质的缓冲溶液中的药物释放行 为，评价了其体外细胞毒性。 该载药纳米凝胶在不含谷胱甘肽浓度的 pH7.4 缓冲液中， 随着纳米凝胶中巯 基含量的增加，累积释药率降低。在模拟血液、正常细胞内以及肿瘤细胞内的 药物释放时，该载药纳米凝胶对肿瘤细胞具有明显的选择性药物释放，由于肿 瘤细胞内高浓度的谷胱甘肽，可使二硫键断开；此外，较低的 pH 值有利于纳米 凝胶疏水性外壳的溶胀，使其内部的二硫键更加完全的暴露在外部高浓度谷胱 甘肽中，从而导致较为快速、完全的药物释放。 关键词：羧甲基壳聚糖，凝胶，纳米凝胶，pH 敏感性，还原响应性
pH/GSH
响 应 羧 甲 基 壳 聚 糖 水 凝 胶 及 纳 米 凝 胶 的 研 究
（申请医学硕士学位论文）
pH/GSH 响应羧甲基壳聚糖水凝胶 及纳米凝胶的研究
培 养 单 位 ： 化学工程学院
高 成
学科专业：药 学 研 究 生 ： 高成 指 导 教 师 ： 殷以华 教授
武 汉 理 工 大 学
2014 年 5 月
万方数据
分类号 UDC
密
来自百度文库
级 10497
学校代码
学
题 目 英 文 题 目 研究生姓名 指导教师 姓名
位
论
文
pH/GSH 响应羧甲基壳聚糖水凝胶及纳米凝胶的研究
The research on the pH/GSH responsive hydrogels and nanogels from carboxymethyl chitosan
学位论文使用授权书
本人完全了解武汉理工大学有关保留、 使用学位论文的规定， 即 学校有权保留并向国家有关部门或机构送交论文的复印件和电子版， 允许论文被查阅和借阅。 本人授权武汉理工大学可以将本学位论文的 全部内容编入有关数据库进行检索， 可以采用影印、 缩印或其他复制 手段保存或汇编本学位论文。 同时授权经武汉理工大学认可的国家有 关机构或论文数据库使用或收录本学位论文， 并向社会公众提供信息 服务。 （保密的论文在解密后应遵守此规定）
III
万方数据
electrostatic attraction between unionized carboxyl group and amino group. The Overshooting effect of hydrogel in pH≤5.0 buffer solution followed the kinetics model proposed by Dí ez-Peňa and so on. When the hydrogel was in pH > 5.0 medium, it swelled until it reaches swelling equilibrium. However, it did not appear overshooting effect in pH ≤ 5.0 buffer solution because of the ionization of carboxyl group, resulting in electrostatic repulsion, and the hydrogen bonds between the adjacent carboxyl disappeared. The swelling kinetics of hydrogel in pH> 5.0 buffer solution of follow Schott secondary swelling kinetics. (3) The pH / GSH response carboxymethyl chitosan nanogel was preparation from chitosan. Their morphology, particle size and distribution were characterized by transmission electron microscopy (TEM) and dynamic light scattering instrument (DLS). Their stability, pH sensitivity and restore reduction responsibility were studied and the formation mechanism of the core-shell nanogels was researched. TEM show that the core-shell structure nanogels had regular shape, uniform distribution, and the average diameter was about 160nm. The DLS results show the particle size and dispersion coefficients of nanogels placed after one month in the pH7.4 buffer solution were not significantly changed, showing that the nanogel had good stability. At lower concentrations (5μM) of glutathione, the average particle size was not change, but a significant increase was observed in 5mM and 20mM glutathione, showing reduction responsibility. The particle size changed with the pH value, exhibited good pH sensitivity, and the sensitivity degree was related to the thiol content in thiolated carboxymethyl chitosan. Amphiphilic macromolecules can self-assemble into nanoparticles in aqueous solution, and crosslink in suit to obtain a core-shell nanogel. The preparation method is simple and does not require the use of a crosslinking agent. The prepared nanogels had pH/redux responsibility. Taking into account differences environment of tumor cells and normal cells, the nanogel could be used for controlled drug delivery to tumor cells. Nanogel has a large amount of carboxyl group at its surface, which could be chemically modified by ligands for tumoru targeted delivery. (4) Taking methotrexate (MTX) as the model drug, the drug release behavior of pH/GSH responsive carboxymethyl chitosan nanogel in simulated media of human blood, normal cells and tumor cell was studied. Its in vitro cytotoxicity was
研究生（签名） ：
导师（签名） ：
日期
万方数据
中 文 摘 要
智能水凝胶和纳米凝胶能够较为迅速的响应外界环境刺激，如 pH、温度、 离子强度、还原电势等。这些特点使它们有望作为药物控释系统的载体，能够 实现对药物的有效负载和控释。在各种环境刺激因素中，pH 敏感性的研究较为 广泛，并且 pH 值在正常细胞和肿瘤细胞中的不同，因而肿瘤细胞内较低的 pH 值常被当做肿瘤细胞药物释放的一种触发因素。二硫键是一种可逆的化学键， 能够稳定存在于人体血液中，在细胞内则被较高浓度谷胱甘肽劈开，然而肿瘤 细胞内谷胱甘肽浓度为正常细胞内的 4 倍，因而在研究肿瘤内药物控释时经常 将二硫键引入到所设计的纳米粒子中。本文以壳聚糖为基质，分别制备了 pH/GSH 响应羧甲基壳聚糖水凝胶和纳米凝胶，并对其性质进行了相关研究，主 要内容和结论如下： （1）以壳聚糖为原料，制得 pH/GSH 响应羧甲基壳聚糖水凝胶，并表征凝 胶的结构和相关物理力学性能，研究凝胶的谷胱甘肽响应性和 pH 敏感性。 制备该凝胶的反应条件温和，易于控制，且在反应过程中，并没有引入巯 基，而是直接引入了二硫键，使得反应无需在无氧条件下进行，大大简化了制 备条件。此外，将 2，2'-二硫代二苯甲酸酰化，使其更容易接枝到羧甲基壳聚糖 上，增加了凝胶中二硫键的含量。 （2）研究了 pH/GSH 响应羧甲基壳聚糖水凝胶的溶胀动力学性质。 凝胶在 pH≤5.0 溶液中出现过溶胀现象， 这是由于凝胶内部网络结构中相邻 羧基之间会产生氢键作用，并且在该 pH 环境下，未电离的羧基和氨基之间也会 有静电吸引作用， 两者共同导致了过溶胀现象的出现。 凝胶在 pH≤5.0 介质中的 过溶胀效应遵循 Dí ez-Peňa 等提出的动力学模型。凝胶在 pH>5.0 的介质中时， 吸水膨胀直至达到溶胀平衡，并没有出现在 pH≤5.0 缓冲溶液中的过溶胀现象， 这主要是由于此时羧基大部分离子化，产生静电排斥作用，并且凝胶内部相邻 羧基的氢键作用也消失，故导致过溶胀现象消失。凝胶在 pH>5.0 缓冲溶液中的 溶胀动力学过程遵循 Schott 二级溶胀动力学方程。 （3）以壳聚糖为基质，制备了 pH/GSH 响应羧甲基壳聚糖纳米凝胶，采用 透射电镜和动态光散射仪对该纳米凝胶的形态、粒径及分布进行表征，并研究 其稳定性、pH 敏感性和还原响应性，探讨该核壳型纳米凝胶的形成机制。 透射电镜和结果显示，该纳米凝胶为核壳结构、形态规整、分布均一，平
高 成 殷 以 华 职称 教 授 学位 博 士
单位名称 武汉理工大学化工学院 邮编 430070 申请学位级别 论文提交日期 硕 士 学科专业名称 论文答辩日期 药 学
学位授予单位 武汉理工大学 学位授予日期 答辩委员会主席 评阅人
2014 年 5 月
万方数据
独 创 性 声 明
本人声明， 所呈交的论文是本人在导师指导下进行的研究工作及 取得的研究成果。 尽我所知， 除了文中特别加以标注和致谢的地方外， 论文中不包含其他人已经发表或撰写过的研究成果， 也不包含为获得 武汉理工大学或其他教育机构的学位或证书而使用过的材料。 与我一 同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说 明并表示了谢意。 签 名： 日 期：
II
万方数据
Abstract
Intelligent hydrogels and nanogels can respond to external environmental stimulus rapidly, such as pH, temperature, ionic strength, redox potential and so on. These features make them expected as a carrier for controlled drug release system to achieve effective drug load and release. In the various environmental stimuli, pH sensitivity was widely studied. The pH values in normal cells and tumor cells are different. Thereby, the lower pH of the tumor cells is often used as a trigger drug release of tumor cells. A reversible disulfide bond can be stable in human blood, while was split in the higher the concentration of intracellular glutathione. As far as we know, the concentration of glutathione in tumor cells is 4 times as that in normal cells. Thus the design of disulfide cross-linked nanoparticles was often introduced into the drug release within the tumor. In this paper, chitosan was chose as a substrate, and the novel pH/GSH responsive carboxymethyl chitosan hydrogel and nanogel were prepared. Their properties were studied, and the main contents and conclusions are as follows: (1) The novel pH/GSH responsive hydrogel was prepared from chitosan. The structures and associated physical and mechanical properties of hydrogel were characterized. There glutathione responsibility and pH sensitivity were studies. The preparation of hydrogel was under mild reaction conditions and easy to control. During the reaction, the thiol group was not introduced, but a disulfide bond was introduced directly. So the reaction could take place successfully without anaerobic conditions, simplifying the preparation conditions. Further, 2,2'-Dithiodibenzoic acid was acylated, making it easier to grafte to the carboxymethyl chitosan. As a result, the content of disulfide bonds in hydrogel was increased. (2) The swelling kinetics of pH/GSH responsive carboxymethyl chitosan hydrogel was investigated. Overshooting effect of hydrogel was appeared in pH≤5.0 buffr solution. This may be attributed to the following two reasons. One is the hydrogen-bond interaction between adjacent carboxyl groups in the network structure of the gel; The other one is