基于X射线成像研究PLGA微球的三维结构及性能

发布时间：2018-06-01 00:36

本文选题：硬X射线显微成像 + 泽尼克相位衬度　；参考：《中国科学技术大学》2014年博士论文

【摘要】：近些年来,伴随着基因工程重组技术和蛋白组学的飞速进步,许多新型多肽、蛋白类药物在治疗、诊断和疫苗预防疾病方面发挥着重要的作用。发展多肽、蛋白类药物的缓释制剂是解决长时间用药、药物毒副作用及耐受性的关键。聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)是一种人工合成的生物可降解高分子载体材料。将药物溶解或分散其中形成的微小球状实体,称之为PLGA微球。PLGA微球具有无毒、生物相容性好和缓控释给药等众多优点,在人体内可缓慢降解,最后形成C02和H20排出体外。现阶段已在人工导管、药物缓控释载体和组织工程等领域广泛使用。当改变PLGA微球的配方或者制备工艺时,PLGA微球的内部结构以及性能会发生变化。但是目前无法定量地分析微球配方、内部结构和性能三者之间的关系,这大大限制了微球制备工艺的改进以及新配方的研制。那么,如何建立PLGA微球配方、内部结构和性能的定量关系模型就成为了缓控释给药载体研究和开发的重点。无损获得PLGA微球内部的孔洞大小、分布和孔隙率等参数就显得非常有意义,但是目前的探测方法(如光学显微镜和电子显微镜)很难满足上述要求。为此,发展一种能够观察孔隙率和孔洞大小、分布等结构信息的三维显微成像工具,原位无损伤研究PLGA微球三维结构及其载药量、包封率等系能参数,对研究具有多孔结构的缓控释给药载体具有普遍的重要意义。近些年来采用波带片放大成像的硬X射线三维成像技术迅速的发展起来。由于硬X射线三维成像技术无需机械切片,就可以得到较厚材料的三维结构数据,而被广泛的应用于众多学科领域。北京同步辐射光源建设了一条基于波带片的硬X射线显微成像光束线站,其成像空间分辨率达到30纳米。在这套大型设备的基础上,本论文主要进行了以下几方面的工作： 1.总结了PLGA微球成像和X射线显微技术。分析了X射线显微成像技术的发展,尤其是北京同步辐射纳米分辨X射线显微成像装置结合泽尼克相位衬度成像和纳米CT新技术在PLGA微球成像上的优势和前景。提出了基于双探测器的X射线微分相位衬度成像新方法,为X射线相位衬度成像的发展提供了新的方向。 2.硬X射线PLGA微球成像的方法学研究。通过调研和实验经验总结,摸索出了适合硬X射线PLGA微球成像的样品处理流程：固定,脱水,重金属染色,干燥。通过这些流程处理的PLGA微球,在BSRF纳米显微成像站大视场模式下,结合泽尼克相位衬度,显示出了明显的衬度,清晰准确的探明了PLGA微球内部的三维结构。同时对比了泽尼克相位衬度成像和吸收成像,阐明了泽尼克相位衬度在硬X射线PLGA微球成像中的显著优势。 3.硬X射线相位衬度成像方法重构PLGA微球的三维结构。利用得到的三维重构片层准确地将微球自身、内部孔洞和孔洞内壁吸附的蛋白区分开来。并在三维空间里提取了一系列的重要结构参数,如孔洞大小,孔洞分布,孔洞体积面积比,孔隙率等,使用光镜或电镜等实验设备无法定量分析获得这些重要的结构参数。这些三维结构参数的获取为我们了解分析PLGA内部的微观结构提供了必不可少的信息。 4.基于硬X射线相位衬度成像分析PLGA微球内部结构和性能之间的关系。利用硬X射线相位衬度成像获得的PLGA微球结构参数和三维结构视图,分析了微球制备时内水相氯化钠浓度对微球结构及性能的影响。内水相氯化钠浓度越高,内外水相的渗透压差越大。在微球完全固化之前,外水相中的水在渗透压的作用下透过有机相进入内水相,使内水相液滴的体积增加,导致固化之后微球孔隙率和孔径的增加,同时由于内水相液滴体积增加,W1/0/W2体系的稳定性降低,内水相液滴容易逃逸至外水相,从而使微球中蛋白质的含量降低。此外,本文还分析了微球内部孔洞大小对微球载药量的影响,为PLGA微球的研发和改进提供了重要的依据。
[Abstract]:In recent years, with the rapid progress of recombinant gene engineering and proteomics, many new peptides, protein drugs play an important role in the treatment, diagnosis and vaccine prevention, and the development of polypeptide and protein drug sustained-release preparation is the key to solve the long time drug use, drug side effects and tolerance.
Poly (lactic acid hydroxy acetic acid) copolymer (poly (lactic-co-glycolic acid), PLGA) is a synthetic biodegradable polymer carrier material. It dissolves or disperses the small spherical substance formed by the drug. It is called the PLGA microsphere.PLGA microspheres with non-toxic, biocompatible and slow controlled release drugs and so on. It can be slowed in human body. At last, C02 and H20 are excreted in vitro. At present, it has been widely used in artificial catheter, drug delivery system, tissue engineering and other fields.
When the formula or preparation process of PLGA microspheres is changed, the internal structure and properties of the PLGA microspheres will change. However, the relationship between the microspheres formula, the internal structure and the three properties of the microspheres can not be quantitatively analyzed at present, which greatly restricts the improvement of the microsphere preparation process and the development of the new ligand. Then, how to establish the PLGA microsphere formula, The quantitative relationship model of internal structure and performance has become the focus of research and development of slow controlled release carrier. It is very meaningful to obtain the parameters of pore size, distribution and porosity within PLGA microspheres, but the current detection methods, such as optical microscopy and electron microscopy, are difficult to meet the requirements mentioned above. A three-dimensional microscopic imaging tool that can observe porosity and pore size, distribution and other structural information. In situ no damage study of the three-dimensional structure of PLGA microspheres and its drug loading, encapsulation efficiency and other parameters. It is of universal importance for the study of the slow controlled release carrier with porous structure. The hard X ray three-dimensional imaging technology has developed rapidly. Because the hard X ray three-dimensional imaging technology does not need mechanical slice, it can get the three-dimensional structure data of the thick material, and is widely used in many subject fields. The Beijing synchrotron radiation source has built a hard X ray microscopic imaging beam line station based on the wave band, its imaging space The resolution is 30 nm. Based on this set of large-scale equipment, this paper mainly focuses on the following aspects:
1. the PLGA microsphere imaging and X ray microscopy were summarized.
The development of X ray microscopy imaging technology is analyzed, especially the advantages and prospects of Beijing synchrotron radiation nanoscale X ray microscopic imaging device combined with Zernike phase contrast imaging and nano CT new technology in PLGA microsphere imaging. A new method of X ray differential phase contrast imaging based on dual detector is proposed, which is a phase contrast of X ray. The development of the image provides a new direction.
A methodological study of 2. hard X ray PLGA microsphere imaging.
Through investigation and experimental experience, we find out the sample processing flow suitable for the hard X ray PLGA microsphere imaging: fixed, dehydrated, heavy metal dyeing and drying. The PLGA microspheres treated by these processes, under the large field mode of the BSRF nanoscale imaging station, combined with the Zernike phase contrast, showed obvious contrast, clear and accurate exploration. The three-dimensional structure of the PLGA microspheres was clarified and the Zernike phase contrast imaging and absorption imaging were compared. The significant advantages of the Zernike phase contrast in the hard X ray PLGA microsphere imaging were clarified.
3. hard X ray phase contrast imaging method reconstructs the three-dimensional structure of PLGA microspheres.
A series of important structural parameters, such as hole size, hole distribution, pore volume area ratio, porosity and so on, can not be quantitatively obtained by using the three-dimensional reconstructed slice layer to distinguish the protein adsorbed in the microsphere itself, the inner hole and the inner wall of the hole. These important structural parameters, the acquisition of these three dimensional structural parameters, provide essential information for us to understand the microstructure of PLGA.
4. based on hard X ray phase contrast imaging, the relationship between the internal structure and properties of PLGA microspheres was analyzed.
The structural parameters of the PLGA microspheres and the three-dimensional structure view obtained by the hard X ray phase contrast imaging were used to analyze the effect of the concentration of sodium chloride on the structure and properties of the microspheres during the preparation of the microspheres. The higher the concentration of sodium chloride in the internal water phase, the greater the osmotic pressure of the internal and external water phase. The volume of the inner water droplet is increased by the use of the organic phase into the internal water phase, which leads to the increase of the porosity and pore size of the microspheres after curing. At the same time, the stability of the W1/0/W2 system decreases because of the increase in the volume of the inner water droplet, and the internal water droplets easily escape to the external water phase, thus reducing the content of the protein in the microspheres. In addition, this paper also divides the water droplets into the microspheres. The effect of pore size on the drug loading of microspheres was analyzed, which provided an important basis for the development and improvement of PLGA microspheres.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08

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