单独剪切制备过氧化氢酶纳米粒的表征及过程模拟研究

发布时间：2018-04-20 21:14

本文选题：过氧化氢酶 + 纳米粒　；参考：《天津大学》2015年硕士论文

【摘要】：过氧化氢酶(CAT)是一种通过催化机体内活性氧自由基的降解来保护细胞免受氧化损伤的蛋白质,在众多疾病治疗中具有重要的应用潜力。但由于其具有一般蛋白药物稳定性差、易失活等特点,限制了临床实践中的应用。因此开发具有对蛋白质结构和活性影响小的制剂工艺成为目前生物制剂领域研究的热点。本文应用单独剪切方法将CAT制备成纳米粒,并对该纳米粒中蛋白质的构象以及活性进行了研究,发现在CAT溶液浓度为0.2 mg/m L、剪切速率217 s-1、剪切时间5 min条件下可制备出粒径为182±8.5 nm、粒度分布均匀、活性保持90%的纳米粒。应用光谱学方法观察发现在该条件下的剪切对CAT构象的影响很小。纳米粒在含有蛋白酶的模拟肠液中孵育4小时,活性仅有20%损失,说明纳米凝聚后的CAT稳定性和抵抗蛋白酶降解的能力都有大幅度的改善。另外,本文应用计算流体力学对单独剪切法制备过程的流场模式,速度场分布,能量耗散分布等流体力学特性进行了数值模拟,发现在大小不同的制备装置中,高速和高能区域都主要集中于搅拌器附近,近壁处和近液面处存在死区,为装置设计改良以将流场均匀分布避免死区等优化措施提供有效信息。通过将粒数平衡与计算流体力学进行耦合,模拟单独剪切制备CAT形成纳米粒的过程及其Sauter平均粒径随时间的演变,在剪切速率设定为217 s-1时,得到的Sauter平均粒径稳定在208 nm,和实验测定的182 nm吻合度较好,同时考察了剪切速率,初始CAT浓度及装置放大对模拟的Sauter平均粒径的影响,为单独剪切法的实际应用奠定了理论基础,并为生产放大提供了有效信息。
[Abstract]:Catalase catalase (CAT) is a kind of protein which can protect cells from oxidative damage by catalyzing the degradation of reactive oxygen free radicals in organism. It has important application potential in the treatment of many diseases. However, because of its poor stability and inactivation of common protein drugs, its application in clinical practice is limited. Therefore, the development of preparation technology with little effect on protein structure and activity has become a hotspot in the field of biological agents. In this paper, CAT nanoparticles were prepared by single shear method, and the conformation and activity of proteins in the nanoparticles were studied. It was found that when the concentration of CAT solution was 0.2 mg/m / L, the shear rate was 217s-1 and the shearing time was 5 min, the particle size was 182 卤8.5 nm, the particle size distribution was uniform, and the activity remained 90%. It is found that shear has little effect on the conformation of CAT under this condition. When the nanoparticles were incubated in the simulated enteric fluid containing protease for 4 hours, the activity was only 20% lost, which indicated that the stability of CAT and the ability of resisting protease degradation were greatly improved after nanocrystalline agglomeration. In addition, computational fluid dynamics is used to simulate the flow field model, velocity field distribution, energy dissipation distribution and other hydrodynamic characteristics of single shear preparation process. The high speed and high energy regions are mainly concentrated near the agitator, and there are dead zones near the wall and near the liquid level, which provides effective information for the design and improvement of the device so as to avoid the dead zone and other optimization measures for uniform distribution of the flow field. By coupling particle number balance with computational fluid dynamics, the process of preparing CAT into nanocrystals by single shear and the evolution of Sauter mean particle size with time were simulated. When the shear rate was set to 217 s ~ (-1), The average particle size of Sauter is stable at 208 nm, which is in good agreement with the experimental results at 182 nm. The effects of shear rate, initial CAT concentration and device amplification on the average particle size of simulated Sauter are also investigated. It lays a theoretical foundation for the practical application of the single shear method and provides effective information for the production amplification.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O629.8;TB383.1

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