研究两亲性聚合物包被的金纳米颗粒与血浆蛋白形成的蛋白晕

发布时间：2018-05-29 03:36

本文选题：蛋白晕 + 两亲性聚合物　；参考：《内蒙古农业大学》2017年博士论文

【摘要】：随着纳米技术的兴起,纳米材料已经广泛应用于各种领域,尤其是生物医药领域中。众所周知,蛋白会与纳米材料表面结合形成蛋白晕,这是一种不可避免的现象。经过这些年的研究,人们对蛋白晕已经有了初步的认识。实际上蛋白晕就是纳米材料作为外源物质进入生物体内以后生物体产生免疫反应的结果。蛋白的构象改变可能会引起它功能的改变;纳米材料的表面性质和功能也会由于材料类型、尺寸、形状、电荷、表面修饰等不同而表现出很大的差异。因此不同的纳米材料与蛋白之间的相互作用会受到多种因素的综合影响。有研究显示蛋白吸附到纳米材料表面上以后其构象发生了改变。蛋白在受到纳米材料影响的同时,也会影响纳米材料。许多研究表明蛋白晕使纳米材料在生物体内的生物学特性、生物分布、细胞毒性等都发生了变化,这些变化会影响纳米材料在生物体内的功能,所以蛋白晕成为纳米材料在生物医药领域应用中的一大研究热点。一般情况下,纳米材料进入体内首先面对的是血液循环系统,血浆中许多蛋白都能够与纳米材料形成蛋白晕,大多数研究也主要是聚焦在血浆蛋白与纳米材料的相互作用上。本论文利用光学技术(吸收光谱、荧光光谱、动态光散射)、离心、尺寸排阻色谱、质谱、凝胶电泳等方法研究了血浆中含量较高的蛋白(牛血清白蛋白、纤维蛋白原、转铁蛋白)与两亲性聚合物包被的金纳米颗粒之间形成的蛋白晕,主要展开了以下工作:1.研究牛血清白蛋白与两亲性聚合物包被的金纳米颗粒形成的离散型蛋白晕。用凝胶电泳的方法可以分离出吸附一个、两个及多个牛血清蛋白的蛋白-纳米颗粒复合物。一般情况下蛋白晕中所含蛋白的具体数目是很难计算的,通常都只是得到蛋白的相对含量。结合吸收光谱与离心法计算出纳米颗粒表面饱和吸附蛋白的数目,结果说明两亲性聚合物包被的金纳米颗粒表面能够吸附的蛋白数目是有限的,这是由纳米颗粒表面性质和大小决定的。这种方法还可以计算不能形成离散型蛋白晕的蛋白(胰凝乳蛋白酶)在纳米颗粒上的数目,最后推导出一个适用于两亲性聚合物包被的纳米材料与蛋白形成的蛋白晕中蛋白数目的计算公式,为以后控制纳米材料上吸附蛋白的数目打下基础。2.测定牛血清白蛋白与两亲性聚合物包被的金纳米颗粒形成的蛋白晕的尺寸。凝胶电泳、动态光散射、尺寸排阻色谱的原理都与被测分子的尺寸相关,因此利用这三种方法能够测定和计算牛血清白蛋白与形成的离散型蛋白晕的尺寸。之前分子模拟的结果已经显示牛血清白蛋白是嵌入到两亲性聚合物中的,所以两亲性聚合物包被的金纳米颗粒吸附了蛋白之后尺寸并不会出现明显的改变。对比三种尺寸计算方法得出的结果,最准确的是尺寸排阻色谱法,其次是动态光散射,误差最大的是凝胶电泳。但每种方法都有各自适用的体系,还是需要根据实际情况来选择合适的计算方法。3.研究牛血清白蛋白与两亲性聚合物包被的金纳米颗粒之间的相互作用力。之前的一系列结果都表明牛血清白蛋白与两亲性聚合物包被的金纳米颗粒形成的蛋白晕非常稳定。利用凝胶电泳和质谱得到了牛血清白蛋白中三条与纳米颗粒结合的多肽片段,长度分别是13、13和15个氨基酸残基,每条片段都含有7个疏水性氨基酸。而聚丙烯酰胺凝胶电泳的结果显示结合位点的多肽片段分子量约为13 kDa,再通过与牛血清白蛋白全部氨基酸序列的比对可以推断出这条多肽片段全长应该是105个氨基酸残基,其中一共有46个疏水性氨基酸残基,并且这些疏水性氨基酸分布比较集中,推测这段多肽的疏水性较强。荧光淬灭法也计算出牛血清白蛋白与两亲性聚合物包被的金纳米颗粒之间的结合常数远大于一般的极限值,温度变化与结合常数呈负相关,说明二者的荧光淬灭模式是静态淬灭,整个反应体系的热力学参数符合热力学第二定律。这些数据都证明了它们之间的相互作用力非常强。吸附的驱动力有很多,但多肽片段的疏水性和两亲性聚合物的疏水链导致了二者吸附的主要驱动力是疏水作用力。4.研究转铁蛋白与两亲性聚合物包被的金纳米颗粒形成的蛋白晕。同样利用凝胶电泳、荧光淬灭法对转铁蛋白与两亲性聚合物包被的金纳米颗粒形成的蛋白晕进行了分析。转铁蛋白的分子量约77kDa,粒径约为3.91nm,与牛血清白蛋白类似,所以转铁蛋白得到了与牛血清白蛋白相似的凝胶电泳结果。同时也发现转铁蛋白能够与纳米颗粒形成离散型蛋白晕。结合常数的大小说明它们之间的结合是非常稳定的。转铁蛋白是癌症治疗中常用的靶向分子,因此研究它与纳米颗粒形成的蛋白晕对于它的靶向应用具有指导意义。5.研究纤维蛋白原与两亲性聚合物包被的金纳米颗粒形成的蛋白晕。纤维蛋白原也属于血浆中的含量较高的蛋白,纤维蛋白构象的改变与一些通路的激活有关。因为纤维蛋白原的分子量和尺寸都非常大,所以它与两亲性聚合物包被的金纳米颗粒形成的蛋白晕明显较牛血清白蛋白和转铁蛋白大,在凝胶电泳上有明显的滞后。荧光淬灭的数据也显示纤维蛋白原与纳米颗粒形成的是硬蛋白晕。纳米颗粒结合一个如此大的分子肯定会对纳米颗粒产生影响,今后还会继续研究。
[Abstract]:With the rise of nanotechnology, nanomaterials have been widely used in various fields, especially in the field of biomedicine. It is well known that protein can be combined with the surface of nanomaterials to form a protein halo. This is an inevitable phenomenon. After these years, people have already had a preliminary understanding of the halo. In fact, the protein halo is the same. Nanomaterials are the result of the immune response of the organism after entering the organism. The conformation change of the protein may cause the change of its function; the surface properties and functions of the nanomaterials also show great differences due to the material type, size, shape, charge, surface modification and so on. The interaction between materials and proteins will be influenced by a variety of factors. Studies have shown that proteins are adsorbed on the surface of nanomaterials to change their conformations. The proteins can also affect nanomaterials while they are affected by nanomaterials. Many studies show that the protein halo makes the nanomaterials biological in the organism, Biological distribution and cytotoxicity have changed, and these changes will affect the function of nanomaterials in the organism. So the protein halo becomes a hot research hotspot in the application of biological medicine. In general, the nano materials are first faced with the blood circulation system, and many proteins in the plasma can be found. Most of the research is mainly focused on the interaction between plasma proteins and nanomaterials. This paper uses optical techniques (absorption spectra, fluorescence spectra, dynamic light scattering), centrifugation, size exclusion chromatography, mass spectrometry, gel electrophoresis and other methods to study the protein (bovine serum albumin) in plasma. Fibrinogen, transferrin, and the protein halo formed between two amphiphilic polymer coated gold nanoparticles. The following work is carried out mainly: 1. to study the discrete protein halo formed by the gold nanoparticles of bovine serum albumin and two amphiphilic polymer clad. A gel electrophoresis method can be used to separate one, two and multiple bovine blood. Albumin nanoparticles complex. In general, the specific number of proteins contained in the protein halo is difficult to calculate. Usually, the relative content of the protein is obtained. The number of the surface saturated adsorbed proteins on the surface of the nanoparticles is calculated by the absorption spectrum and centrifugation. The results indicate that the gold nanoparticles of the two amphiphilic polymer clad are shown. The number of proteins that can be adsorbed on the surface is limited, depending on the surface properties and size of the nanoparticles. This method can also calculate the number of proteins that can not form a discrete type of protein halo (chymotrypsin) on the nanoparticles. Finally, it derives an egg that is suitable for the formation of the two amphiphilic polymer coated nano materials and protein. The calculation formula of the number of protein in the white halo to determine the size of the protein halo formed by the determination of the number of adsorbed proteins on the nanomaterials by.2.. The principle of gel electrophoresis, dynamic light scattering, and size exclusion chromatography are all related to the size of the molecules. These three methods can determine and calculate the size of the BSA and the formed discrete protein halo. The results of the previous molecular simulation have shown that the bovine serum albumin is embedded in the two amphiphilic polymer, so the size of the gold nanoparticles covered by the two amphiphilic polymer clad is not significantly changed after the egg white. Compared to the results obtained by three methods of size calculation, the most accurate is size exclusion chromatography, followed by dynamic light scattering, and the maximum error is gel electrophoresis. But each method has its own system, or it is necessary to choose the appropriate method based on the actual situation,.3., to study the bovine serum albumin and the two amphiphilic polymer package. The interaction between gold nanoparticles. A series of previous results showed that the halo of bovine serum albumin and the gold nanoparticles of the two amphiphilic polymer coated gold nanoparticles were very stable. By gel electrophoresis and mass spectrometry, three peptide fragments of bovine serum albumin combined with nanoparticles were obtained, with the length of 13,13 and 15 ammonia, respectively. Each fragment contains 7 hydrophobic amino acids, and the results of polyacrylamide gel electrophoresis show that the molecular weight of the polypeptide fragment at the binding site is about 13 kDa, and the total length of this polypeptide fragment should be 105 amino acid residues by comparison with all the amino acid sequences of bovine serum albumin, with a total of 46 of them. The hydrophobic amino acid residues, and the distribution of these hydrophobic amino acids are concentrated, speculate that the polypeptide has a strong hydrophobicity. The fluorescence quenching method also calculated that the binding constant between the bovine serum albumin and the gold nanoparticles of the two amphiphilic polymer package is far greater than the general limit value, and the temperature change is negatively correlated with the binding constant. The fluorescence quenching mode of the two is statically quenched and the thermodynamic parameters of the whole reaction system conform to the second law of thermodynamics. These data prove that the interaction force between them is very strong. There are many driving forces of the adsorption, but the hydrophobicity of the polypeptide fragment and the hydrophobic chain of the two parent polymer lead to the main driving force of the two adsorption. It is a hydrophobic interaction force.4. to study the protein halo formed by the gold nanoparticles of the transferrin and two amphiphilic polymer coated gold nanoparticles. The gel electrophoresis and fluorescence quenching method are used to analyze the halo of the gold nanoparticles formed by the transferrin and the two amphiphilic polymer coated gold nanoparticles. The molecular weight of the transferrin is about 77kDa, the particle size is about 3.91nm, and the bovine blood. The albumin is similar, so the transferrin obtained the gel electrophoresis results similar to the bovine serum albumin. It also found that the transferrin could form a discrete protein halo with the nanoparticles. The binding constant of the binding constant indicates that the binding between them is very stable. The protein halo formed with the nanoparticles has a guiding significance for its targeting application.5. to study the protein halo formed by fibrinogen and gold nanoparticles containing two amphiphilic polymer packages. Fibrinogen is also a protein in the plasma, and the changes in the conformation of fibrin are related to the activation of some pathways. The molecular weight and size of the white source are very large, so the protein halo formed by the gold nanoparticles in the two amphiphilic polymer package is obviously larger than that of bovine serum albumin and transferrin, and there is a significant lag in gel electrophoresis. The fluorescence quenching data also show that the fibrinogen and nanoparticles form a hard protein halo. Such a large molecule will definitely affect the nanoparticles and will continue to study in the future.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08

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