多功能氧化石墨烯基因载体的制备及其靶向治疗的初步研究

发布时间：2018-06-04 12:05

本文选题：氧化石墨烯（GO） + 聚乙烯亚胺（PEI）　；参考：《吉林大学》2015年硕士论文

【摘要】：基因治疗对于治疗疾病相关，尤其是癌症是一个热点。基因治疗最关键的问题就是发展一个合适、高效的基因载体，基因载体可分为病毒型载体和非病毒型载体。病毒型载体由于毒性高和自身的局限性而使用的少，非病毒载体因为安全性高、有效性强得到了广泛的应用，例如：聚合物、脂质体、纳米粒子等等。近几年，非病毒基因载体虽然已经获得了很大的进步，但有些问题包括非特异性靶向位点、毒性和生物稳定等问题依然没有得到很好的解决。因此发展一个有效的纳米载体仍然是基因治疗中最大的挑战。石墨烯，拥有一个碳原子厚的二维层状结构，因为这个特异性在许多领域中都得到了广泛的应用，例如电化学装置，生物应用等等。氧化石墨烯(GO)是石墨烯的水溶性衍生物，由于其容易制备，在水溶液和生理环境中有较好的分散性，好的胶体稳定性已经吸引了很大的兴趣，并且在生物传感器，基因和药物传递，以及在体内外的生物成像等生物领域中也显示了潜在的应用。在本文中，我们以氧化石墨烯为基础，采用对其进行化学偶联修饰，设计一种新型的多功能氧化石墨烯的基因载体GO-PEI-PEG-FA/si-Stat3。我们选择常用的聚合物支化的聚乙烯亚胺（PEI）与羧基化的氧化石墨烯通过偶氮化学反应进行连接，目的是为提高转染效率。引入了聚乙二醇（PEG），为提高溶液的生物稳定性，最后引入一种小分子叶酸（FA），对其进行靶向性修饰，实现靶向治疗的目的。再与si-Stat3质粒通过静电相互作用与GO-PEI-PEG-FA连接。最终形成GO-PEI-PEG-FA/si-Stat3基因载体。我们研究了通过调节不同比例的PEI对GO-PEI-PEG-FA的结构的影响，并在这个过程进一步研究结构对粒径和细胞转染效率的关系，建立了相应的构效关系。结果表明通过采用不同比例的PEI，得到不同接枝量对结构、粒径和稳定性的影响。当接枝量小时，粒径较大，PEI与PEI之间的作用力小，纳米粒子为舒展的状态，并形成了插层的结构，相对应的稳定性则下降；当接枝量逐渐增加粒径却逐渐变小，PEI与PEI之间的作用力增大，纳米粒子为卷曲的状态，形成了剥离的结构，，稳定性也由于作用力的作用增强；当接枝量逐渐增加的缓慢，粒径也几乎趋于平稳，作用力之间达到恒定，稳定性达到最好。我们制备了结构可控，粒径小，稳定性好的基因载体。同时GO-PEI-PEG-FA/si-Stat3复合物沉默了Stat3的表达使达到了靶向治疗肝癌的目的。
[Abstract]:Gene therapy is a hot spot in the treatment of diseases, especially cancer. The key problem of gene therapy is to develop a suitable and efficient gene vector, which can be divided into viral vector and non-viral vector. Viral carriers are less used because of their high toxicity and limitations. Non-viral carriers have been widely used because of their high safety and effectiveness, such as polymers, liposomes, nanoparticles and so on. In recent years, although non-viral gene vectors have made great progress, some problems, such as non-specific targeting sites, toxicity and biological stability, have not been solved. Therefore, the development of an effective nano-carrier remains the biggest challenge in gene therapy. Graphene, which has a two-dimensional layered structure with thick carbon atoms, has been widely used in many fields, such as electrochemical devices, biological applications and so on. Graphene oxide (GOO) is a water-soluble derivative of graphene. Because of its easy preparation, good dispersion in aqueous solution and physiological environment, good colloid stability has attracted great interest, and has attracted great interest in biosensors. Gene and drug delivery, as well as biological fields such as imaging in vivo and in vitro, have also shown potential applications. In this paper, based on graphene oxide, a novel multifunctional graphene oxide gene vector GO-PEI-PEG-FA-FA-Stat3 was designed by chemical coupling modification. In order to improve the transfection efficiency, we selected the commonly used polymer branched polyethyleneimine (PEI) and carboxylated graphene oxide by azo chemical reaction. In order to improve the biological stability of the solution, a small molecule of folate, FAA, was introduced and modified to achieve the purpose of targeted therapy. The GO-PEI-PEG-FA was connected with si-Stat3 plasmid by electrostatic interaction. Finally, the GO-PEI-PEG-FA/si-Stat3 gene vector was formed. We studied the effect of different proportion of PEI on the structure of GO-PEI-PEG-FA, and further studied the relationship of structure to particle size and cell transfection efficiency during this process, and established the corresponding structure-activity relationship. The results show that the effects of grafting amount on structure, particle size and stability are obtained by using different proportion of PEI. When the grafting amount is small, the interaction force between the larger particle size and the PEI is small, and the nanoparticles are in a stretch state, and the intercalation structure is formed, and the corresponding stability decreases. When the grafted amount increases gradually but the interaction force between PEI and PEI increases gradually, the nanoparticles are crimped, and the structure of peeling is formed, and the stability is enhanced because of the effect of the acting force, and when the grafting amount increases slowly, The particle size is almost stable, the interaction force is constant, and the stability is the best. We have prepared gene vectors with controllable structure, small particle size and good stability. At the same time, the GO-PEI-PEG-FA/si-Stat3 complex silenced the expression of Stat3, so that the target therapy of liver cancer was achieved.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450;O613.71

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