靶向性混合纳米胶束的制备及其体内外抗炎效果的研究

发布时间：2018-02-22 12:17

本文关键词： 聚唾液酸胶束类风湿性关节炎地塞米松叶酸胆固醇　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：类风湿性关节炎是一种慢性、免疫系统紊乱性疾病,发病率约占人群的1%。药物的给药系统有效的解决了许多药物低效和高毒的缺点。纳米尺寸的药物给药系统由于能够选择性的聚集在病变部位而成为目前研究的热点,天然多糖是无毒、可生物降解的大分子生物材料,可用于制备纳米尺寸的药物载体。聚唾液酸(PSA)是具有强亲水性的多糖,可以减少网状内皮系统对纳米载体的摄取并延长药物在体内的循环时间。胆固醇是细胞膜的一个组成部分,有利于纳米载体的内吞作用,充当疏水基团。本研究设计和合成了基于PSA-CC的纳米混合胶束,用于改善抗炎药物的递送,提高药效和减小毒性。通过NMR,IR,Size,ζ电位和透射电子显微镜证实了混合胶束合成成功。基于PSA的普通胶束和靶向混合胶束通过自组装形成胶束的临界胶束浓度分别为46.2±3.9μg/ml、32.1±5.2μg/ml。经典抗炎药物地塞米松通过疏水相互作用有效地加载到胶束中,地塞米松的载入使靶向混合纳米胶束的粒径从83.8±13.4nm增加到115.0±12.2nm,纳米胶束粒径有利于药物递送到炎症组织。用巨噬细胞(RAW264.7)进行体外实验,首先,用CCK-8法测定和评价胶束的体外细胞毒性,结果表明制备的载体在所用浓度下无毒。然后,脂多糖(LPS)刺激巨噬细胞,测定促炎因子如肿瘤坏死因子(TNF-α)和白细胞介素-6(IL-6)的细胞表达,评价载药普通胶束和混合胶束的体外抗炎效果,结果表明,装载地塞米松的混合胶束具有比游离地塞米松和载药普通胶束更好的抗炎效果。最后,巨噬细胞与游离香豆素或胶束-香豆素共孵育,通过流式细胞术和荧光显微镜研究胶束的细胞摄取,表明混合胶束具有更强的细胞内吞作用。对小鼠尾根部皮下注射完全弗氏佐剂建立体内关节炎症模型,每天给药,通过测量小鼠足跖厚度、小鼠关节炎指数评分和关节的HE染色切片评价载药体系的体内抗炎效果,结果表明载药胶束能够明显的改善小鼠关节的红、肿现象,足跖厚度减少。用近红外荧光标记载体,研究载体在炎症小鼠体内的分布情况,小动物活体成像结果显示载体能够靶向于炎症部位,且荧光强度能够持续24h。我们又研究了药物在体内的代谢情况,用PK-solver处理数据,游离地塞米松原料药的半衰期为3.49h,载药普通胶束、载药混合胶束药物的半衰期分别为16.52h、19.29h,结果表明载药胶束延长地塞米松在体内的循环时间。综上,本研究构建的靶向载药胶束增强了药物的体外细胞摄取,提高了药物的体内和体外抗炎效果,具有较高的安全性,有望更加安全、有效地治疗类风湿性关节炎。
[Abstract]:Rheumatoid arthritis is a chronic, immune disorder. The incidence of disease is about 1% of the population. The drug delivery system effectively solves the disadvantages of low efficiency and high toxicity of many drugs. Nanoscale drug delivery systems have become a hot research area because of their ability to selectively concentrate in the diseased areas. Natural polysaccharides are nontoxic and biodegradable macromolecular biomaterials, which can be used to prepare nano-sized drug carriers. Polysialic acid (PSAs) is a strong hydrophilic polysaccharide. Cholesterol is an integral part of the cell membrane and is beneficial to the endocytosis of the nano-carrier, which can reduce the uptake of the nanoparticles by the reticuloendothelial system and prolong the circulation time of the drug in the body. In order to improve the delivery of anti-inflammatory drugs, a nanocomposite micelle based on PSA-CC was designed and synthesized to act as a hydrophobic group. The synthesis of mixed micelles was confirmed by NMR-IR IR Size, 味 potential and transmission electron microscope. The critical micelle concentrations of ordinary micelles and targeted mixed micelles based on PSA were 46.2 卤3.9 渭 g / ml / ml and 32.1 卤5.2 渭 g / ml / ml, respectively. The classic anti-inflammatory drug dexamethasone is effectively loaded into micelles through hydrophobic interactions. The loading of dexamethasone increased the particle size of targeted mixed micelles from 83.8 卤13.4 nm to 115.0 卤12.2 nm. The cytotoxicity of micelle in vitro was determined and evaluated by CCK-8. The results showed that the carrier was not toxic at the concentration used. Then, lipopolysaccharide (LPS) stimulated macrophages, and the expression of proinflammatory factors such as tumor necrosis factor TNF- 伪 (TNF- 伪) and interleukin-6 (IL-6) were measured. The anti-inflammatory effect of common micelles and mixed micelles loaded with Dexamethasone was evaluated in vitro. The results showed that the mixed micelles loaded with dexamethasone had better anti-inflammatory effect than free dexamethasone and drug-loaded micelles. Macrophages were co-incubated with free coumarin or micella-coumarin, and the cellular uptake of micelles was studied by flow cytometry and fluorescence microscopy. The results showed that the mixed micelles had stronger endocytosis. A model of arthritis in vivo was established by subcutaneous injection of complete Freund's adjuvant to the tail root of mice. Mouse arthritis index score and HE staining section of joint were used to evaluate the anti-inflammatory effect of drug delivery system in vivo. The results showed that the micelles could obviously improve the redness and swelling of mouse joints and decrease the thickness of feet and plantar. In vivo imaging of small animals showed that the vector could target the inflammatory site and the fluorescence intensity could last 24 h. We also studied the metabolism of drugs in vivo and processed the data with PK-solver. The half-life of free dexamethasone raw drug was 3.49 h, the half life of drug carrying common micelle and drug mixed micelle was 16.52 h or 19.29 h respectively. The results showed that the drug carrier micelle prolonged the circulation time of dexamethasone in vivo. In this study, the targeted drug carrier micelle enhanced the drug uptake in vitro, improved the anti-inflammatory effect of drugs in vivo and in vitro, and has a higher safety, which is expected to be more safe and effective in the treatment of rheumatoid arthritis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

【相似文献】