本文選題：硬X射線顯微成像 + 澤尼克相位襯度�。� 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2014年博士論文

【摘要】：近些年來(lái),伴隨著基因工程重組技術(shù)和蛋白組學(xué)的飛速進(jìn)步,許多新型多肽、蛋白類藥物在治療、診斷和疫苗預(yù)防疾病方面發(fā)揮著重要的作用。發(fā)展多肽、蛋白類藥物的緩釋制劑是解決長(zhǎng)時(shí)間用藥、藥物毒副作用及耐受性的關(guān)鍵。 聚乳酸-羥基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)是一種人工合成的生物可降解高分子載體材料。將藥物溶解或分散其中形成的微小球狀實(shí)體,稱之為PLGA微球。PLGA微球具有無(wú)毒、生物相容性好和緩控釋給藥等眾多優(yōu)點(diǎn),在人體內(nèi)可緩慢降解,最后形成C02和H20排出體外�，F(xiàn)階段已在人工導(dǎo)管、藥物緩控釋載體和組織工程等領(lǐng)域廣泛使用。 當(dāng)改變PLGA微球的配方或者制備工藝時(shí),PLGA微球的內(nèi)部結(jié)構(gòu)以及性能會(huì)發(fā)生變化。但是目前無(wú)法定量地分析微球配方、內(nèi)部結(jié)構(gòu)和性能三者之間的關(guān)系,這大大限制了微球制備工藝的改進(jìn)以及新配方的研制。那么,如何建立PLGA微球配方、內(nèi)部結(jié)構(gòu)和性能的定量關(guān)系模型就成為了緩控釋給藥載體研究和開(kāi)發(fā)的重點(diǎn)。無(wú)損獲得PLGA微球內(nèi)部的孔洞大小、分布和孔隙率等參數(shù)就顯得非常有意義,但是目前的探測(cè)方法(如光學(xué)顯微鏡和電子顯微鏡)很難滿足上述要求。為此,發(fā)展一種能夠觀察孔隙率和孔洞大小、分布等結(jié)構(gòu)信息的三維顯微成像工具,原位無(wú)損傷研究PLGA微球三維結(jié)構(gòu)及其載藥量、包封率等系能參數(shù),對(duì)研究具有多孔結(jié)構(gòu)的緩控釋給藥載體具有普遍的重要意義。近些年來(lái)采用波帶片放大成像的硬X射線三維成像技術(shù)迅速的發(fā)展起來(lái)。由于硬X射線三維成像技術(shù)無(wú)需機(jī)械切片,就可以得到較厚材料的三維結(jié)構(gòu)數(shù)據(jù),而被廣泛的應(yīng)用于眾多學(xué)科領(lǐng)域。北京同步輻射光源建設(shè)了一條基于波帶片的硬X射線顯微成像光束線站,其成像空間分辨率達(dá)到30納米。在這套大型設(shè)備的基礎(chǔ)上,本論文主要進(jìn)行了以下幾方面的工作： 1.總結(jié)了PLGA微球成像和X射線顯微技術(shù)。 分析了X射線顯微成像技術(shù)的發(fā)展,尤其是北京同步輻射納米分辨X射線顯微成像裝置結(jié)合澤尼克相位襯度成像和納米CT新技術(shù)在PLGA微球成像上的優(yōu)勢(shì)和前景。提出了基于雙探測(cè)器的X射線微分相位襯度成像新方法,為X射線相位襯度成像的發(fā)展提供了新的方向。 2.硬X射線PLGA微球成像的方法學(xué)研究。 通過(guò)調(diào)研和實(shí)驗(yàn)經(jīng)驗(yàn)總結(jié),摸索出了適合硬X射線PLGA微球成像的樣品處理流程：固定,脫水,重金屬染色,干燥。通過(guò)這些流程處理的PLGA微球,在BSRF納米顯微成像站大視場(chǎng)模式下,結(jié)合澤尼克相位襯度,顯示出了明顯的襯度,清晰準(zhǔn)確的探明了PLGA微球內(nèi)部的三維結(jié)構(gòu)。同時(shí)對(duì)比了澤尼克相位襯度成像和吸收成像,闡明了澤尼克相位襯度在硬X射線PLGA微球成像中的顯著優(yōu)勢(shì)。 3.硬X射線相位襯度成像方法重構(gòu)PLGA微球的三維結(jié)構(gòu)。 利用得到的三維重構(gòu)片層準(zhǔn)確地將微球自身、內(nèi)部孔洞和孔洞內(nèi)壁吸附的蛋白區(qū)分開(kāi)來(lái)。并在三維空間里提取了一系列的重要結(jié)構(gòu)參數(shù),如孔洞大小,孔洞分布,孔洞體積面積比,孔隙率等,使用光鏡或電鏡等實(shí)驗(yàn)設(shè)備無(wú)法定量分析獲得這些重要的結(jié)構(gòu)參數(shù)。這些三維結(jié)構(gòu)參數(shù)的獲取為我們了解分析PLGA內(nèi)部的微觀結(jié)構(gòu)提供了必不可少的信息。 4.基于硬X射線相位襯度成像分析PLGA微球內(nèi)部結(jié)構(gòu)和性能之間的關(guān)系。 利用硬X射線相位襯度成像獲得的PLGA微球結(jié)構(gòu)參數(shù)和三維結(jié)構(gòu)視圖,分析了微球制備時(shí)內(nèi)水相氯化鈉濃度對(duì)微球結(jié)構(gòu)及性能的影響。內(nèi)水相氯化鈉濃度越高,內(nèi)外水相的滲透壓差越大。在微球完全固化之前,外水相中的水在滲透壓的作用下透過(guò)有機(jī)相進(jìn)入內(nèi)水相,使內(nèi)水相液滴的體積增加,導(dǎo)致固化之后微球孔隙率和孔徑的增加,同時(shí)由于內(nèi)水相液滴體積增加,W1/0/W2體系的穩(wěn)定性降低,內(nèi)水相液滴容易逃逸至外水相,從而使微球中蛋白質(zhì)的含量降低。此外,本文還分析了微球內(nèi)部孔洞大小對(duì)微球載藥量的影響,為PLGA微球的研發(fā)和改進(jìn)提供了重要的依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08

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本文編號(hào)：1962085