磷酸钙纳米颗粒作为蛋白质载体的研究

发布时间：2018-02-07 17:12

本文关键词： 磷酸钙纳米颗粒蛋白质载体 GFP KillerRed　出处：《中国科学技术大学》2015年硕士论文　论文类型：学位论文

【摘要】：蛋白质能催化生化反应,组成受体和细胞膜通道,提供细胞骨架支持,在细胞内转运分子,是体内功能最多样化的生物大分子。蛋白质药物广泛的应用于癌症治疗、疫苗、再生药物、治疗基因缺陷疾病和成像等领域。然而,由于蛋白质特有的性质,比如较大的体积、不同的表面电势、易受损伤的三级结构,将目标蛋白运载入细胞内有很多阻碍。所以蛋白质运载策略中最大的挑战是在不破坏蛋白质结构和生物活性的前提下将蛋白运载到细胞质。大多数蛋白质运载体系依赖于细胞的内吞作用。因此,被运载的目标蛋白很容易在内体／溶酶体弱酸性的采环境中被降解,进而无法到达目标的亚细胞区域发挥作用。基于以上原因,我们用了一种pH敏感的纳米生物材料,来避免目标蛋白在内体／溶酶体中降解,并有效的释放到细胞质中。磷酸钙纳米颗粒是pH敏感的蛋白质运载体系,有很好的生物相容性和生物降解性。在本论文中,我们以磷酸钙纳米颗粒为基础建立了一种有效的细胞内蛋白质运载体系。我们设计了一种简单低成本的磷酸钙纳米颗粒制备方法,检测了纳米颗粒各方面的性质,包括颗粒粒径、颗粒形态、载药率、包封率、pH敏感性质和稳定性。磷酸钙纳米颗粒有很好的分散性并保持稳定的粒径。在内体／溶酶体的弱酸性环境中,蛋白能很快从颗粒上释放,而在生理环境(pH=7.4)中能保持稳定。然后,我们进一步研究了磷酸钙纳米颗粒的细胞摄取、蛋白在细胞内的释放情况以及颗粒上负载蛋白的生物活性。一方面,我们通过荧光显微镜和流式细胞术来研究磷酸钙纳米颗粒的细胞摄取情况；结果表明颗粒能成功有效的被细胞摄取。由于内体／溶酶体逃逸对于细胞内药物的释放是一个阻碍,我们通过激光共聚焦显微镜观察蛋白在细胞内的释放情况：结果表明蛋白质能成功释放到细胞质中。另一方面,SDS-PAGE实验和圆二色谱分析结果表明由于温和的制备方法,磷酸钙纳米颗粒上释放的蛋白能保持其结构不被破坏。另外,磷酸钙纳米颗粒上释放的KillerRed蛋白保留了其生物活性。以上的实验结果表明磷酸钙纳米颗粒是一种合适的蛋白质载体。
[Abstract]:Proteins catalyze biochemical reactions, form receptors and cell membrane channels, provide cytoskeleton support, transport molecules within cells, and are the most versatile biological macromolecules in the body. Regenerative drugs, treatment of genetic defects, imaging, etc. However, because of the specific properties of proteins, such as large volume, different surface potential, vulnerable tertiary structure, There are many obstacles to carrying the target protein into the cell. So the biggest challenge in the protein delivery strategy is to transport the protein to the cytoplasm without destroying the protein structure and biological activity. Most protein carriers. The lineage depends on the endocytosis of the cell. The target protein is easily degraded in a weakly acidic inner / lysosomal environment, which can't reach the target subcellular area. For these reasons, we use a pH sensitive nanomaterials. In order to avoid degradation of target protein in inner body / lysosome and release it into cytoplasm effectively. Calcium phosphate nanoparticles are a pH sensitive protein delivery system with good biocompatibility and biodegradability. We have established an effective intracellular protein delivery system based on calcium phosphate nanoparticles. We have designed a simple and low-cost method for preparing calcium phosphate nanoparticles and examined their properties. Including particle size, particle morphology, drug loading rate, entrapment efficiency, pH sensitivity and stability. Calcium phosphate nanoparticles have good dispersion and maintain stable particle size. The protein was released quickly from the particles and remained stable in the physiological environment (pH = 7.4). Then we further studied the cellular uptake of calcium phosphate nanoparticles. On the one hand, we studied the uptake of calcium phosphate nanoparticles by fluorescence microscopy and flow cytometry. The results showed that the granules were successfully and effectively ingested by the cells, because the escape of the inner body / lysosome was an obstacle to the release of the drugs in the cell. We observed the release of proteins in cells by laser confocal microscopy: the results showed that proteins could be successfully released into the cytoplasm. On the other hand, SDS-PAGE and circular dichroism analysis showed that due to mild preparation methods, The protein released on the calcium phosphate nanoparticles can keep its structure intact. The KillerRed protein released on the calcium phosphate nanoparticles retains its biological activity. The above results show that the calcium phosphate nanoparticles are a suitable protein carrier.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O614.231;TB383.1

【共引文献】