IgG修饰的刺槐素脂质体的研究

发布时间：2018-06-30 21:44

本文选题：刺槐素 + 血脑屏障　；参考：《厦门大学》2014年硕士论文

【摘要】：脑胶质瘤为起源于神经胶质细胞的肿瘤,是最常见的原发性颅内肿瘤。尤其是多形性脑胶质瘤(GBM),它是一类较频繁发生的中枢神经系统性恶性肿瘤,较容易扩散到周围的正常组织。传统的手术治疗很难将脑胶质瘤全部切除,而且很容易复发。目前,最有效的治疗脑胶质瘤的方法是外科手术结合化疗,但是化疗治疗效果非常有限,还会造成系统的许多副作用。这些副作用主要是由于存在血脑屏障(BBB),不允许98%的小分子和100%的大分子通过,化疗药物很难通过BBB,而积累在周围组织产生副作用。因此我们应当设计一种药物载体来传递化疗药物,使其通过血脑屏障并靶向于肿瘤病灶部位。低密度脂蛋白受体相关蛋白(LRP)是存在于BBB上的一种受体,IgG被LRP识别,并通过LRP调节的内吞途径通过血脑屏障。选择刺槐素作为模型药物。刺槐素难溶于水,近几年越来越多的研究表明刺槐素具有保护神经和抗肿瘤的作用。因此我们以脂质体作为载体来传递刺槐素到脑胶质瘤所在部位。以IgG作为靶向材料,引导脂质体通过BBB并靶向于脑胶质瘤U87 MG细胞。这一新型的脂质体可以被用来作为抗脑胶质瘤药物的理想剂型,同时也可作为其他中枢神经系统疾病治疗药物的传递载体。本文研究的主要内容包括以下几个方面:1.合成了 CL-PEG2000-Mal,活化的IgG蛋白与其结合,制备的主动靶向脂质体通过LRP调节的细胞内吞途径进入细胞。2.建立了刺槐素的体外高效液相分析方法和刺槐素脂质体包封率的测定方法(微孔滤膜过滤分离-HPLC检测药物含量),薄膜分散法制备脂质体,挤出、微孔滤膜过滤达到分离游离药物和整粒的目的。刺槐素脂质体的最佳处方为磷脂的含量5%(w/v),总磷脂与胆固醇之比为4:1,药物与磷脂之比为1:20,水化介质为10%海藻糖。IgG修饰的刺槐素脂质体的包封率大于80%,平均粒径为163 nm,PDI为0.352,IgG修饰的脂质体的形状为规则的类球形,拥有良好的体外释放行为。3.MTT实验和细胞摄取实验证明了 IgG修饰的空白脂质体低毒而且能够明显增加U87 MG细胞和bEnd.3细胞对脂质体的摄取。在MTT实验中,与普通刺槐素脂质体和游离刺槐素相比,IgG修饰的刺槐素脂质体对U87 MG细胞增殖的抑制作用最强。在细胞的摄取实验中,U87MG细胞对IgG修饰的脂质体的摄取指数分别是对照组1.22、1.91、2.20、2.27、2.55倍,bEnd.3细胞对脂质体的摄取效果与U87 MG细胞相似。4.转移通过血脑屏障能力实验和体外双靶向性实验都表明了 IgG修饰的脂质体可以高效的通过BBB,靶向于U87MG细胞,而且通过BBB后的脂质体维持着完整性。总之,在本研究的体外的共培养模式中,IgG修饰的脂质体不仅增加了其通过BBB的能力还能够连续靶向于U87 MG细胞。总之,IgG修饰的刺槐素脂质体的MTT实验证实了 IgG球蛋白修饰提高了刺槐素的细胞毒性,U87 MG细胞和bEnd.3细胞的摄取实验、抑制性试验都证明IgG的修饰提高了脂质体的摄取量,而且具有一定的靶向性。脂质体的透过能力试验和体外双靶向性实验与预期实验结果相同,达到既能够通过血脑屏障又能够靶向U87MG细胞,起到连续靶向性的效果。IgG的修饰提高了脂质体的脑靶向性,具有良好的临床应用前景。
[Abstract]:Glioma, the most common primary intracranial tumor, is the most common primary intracranial tumor, especially the GBM. It is a kind of more frequent central nervous system malignant tumor, which is easy to spread to normal tissues around. It is difficult to remove all the glioma by traditional surgical treatment, and it is very easy. Recrudesce. Currently, the most effective treatment of glioma is surgery combined with chemotherapy, but the effect of chemotherapy is very limited and can cause many side effects of the system. These side effects are mainly due to the existence of the blood brain barrier (BBB), which does not allow 98% small molecules and 100% large molecules to pass through, and the chemotherapeutic drugs are difficult to accumulate through BBB. The surrounding tissue produces side effects. Therefore, we should design a drug carrier to pass the chemotherapeutic agents through the blood brain barrier and target the site of the tumor. Low density lipoprotein receptor related protein (LRP) is a receptor on BBB, IgG is identified by LRP, and through the LRP regulated endocytic pathway through the blood brain barrier. Select spines. Sophotin is a model drug. Robinia is difficult to dissolve in water. In recent years, more and more studies have shown that Robinia has a protective effect on nerve and antitumor. Therefore, we use liposome as a carrier to transfer Robinia to the location of brain glioma. IgG is used as a target material to guide liposomes through BBB and target glioma U87 MG cells. This new liposome can be used as an ideal form of anti glioma drugs, and can also be used as a carrier for other central nervous system diseases. The main contents of this study include the following aspects: 1. synthesis of active IgG proteins and the active targeting liposomes are synthesized by CL-PEG2000-Mal. The high performance liquid phase analysis method of Robinia in vitro and the method for determining the encapsulation efficiency of Robinia Robinia liposome were established by the LRP regulated endocytic pathway into the cell.2., and the liposomes were prepared by the microporous filter membrane filtration, and the liposomes were prepared by the thin film dispersion method. The purpose of separating free drugs and whole particles was achieved by extrusion and microporous filtration membrane filtration. The best prescription of sophiotin liposome is 5% (w/v), the ratio of total phospholipid to cholesterol is 4:1, the ratio of drug to phospholipid is 1:20. The encapsulation efficiency of Sophora japonica liposomes modified by 10% trehalose.IgG is more than 80%, the average particle size is 163 nm, PDI is 0.352, the shape of IgG trimming liposome is a regular spherical shape, and has good shape. In vitro release behavior.3.MTT and cell uptake experiments demonstrate that IgG modified blank liposomes are low toxic and can significantly increase the uptake of liposomes by U87 MG cells and bEnd.3 cells. In MTT experiments, the inhibition of IgG modified Robinia liposomes on the proliferation of U87 MG cells in comparison with the normal Robinia liposomes and free Robinia In the cell uptake experiments, the uptake of IgG modified liposomes by U87MG cells was 1.22,1.91,2.20,2.27,2.55 times of the control group. The effect of bEnd.3 cells on the liposomes was similar to that of U87 MG cells,.4. transfer through the blood brain barrier ability experiment and in vitro double targeting experiment all showed IgG modified liposomes. It is possible to efficiently target U87MG cells through BBB, and maintain integrity through the liposomes after BBB. In a word, in the co culture model of this study, IgG modified liposomes not only increase their ability to pass through BBB but also target U87 MG cells continuously. In a word, the MTT experiment of the IgG modified Robinia liposomes confirms I. GG globulin modification improves the cytotoxicity of Robinia, the uptake of U87 MG cells and bEnd.3 cells, and the inhibition test shows that the modification of IgG improves the uptake of liposomes and has a certain target. The permeability test of liposomes and the double targeting experiment in vitro are the same as the expected experimental results. The blood-brain barrier can also target U87MG cells and play a continuous targeting role. The modification of.IgG improves the targeting ability of liposomes and has good clinical application prospects.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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