功能化金纳米颗粒的合成、表征及其与蛋白质和细胞的相互作用

发布时间：2018-04-14 11:17

本文选题：金纳米颗粒 + 表面电荷密度　；参考：《山东大学》2013年博士论文

【摘要】：纳米材料具有潜在医学价值,可以用于载药、载基因等,作为治疗试剂,也可以作为造影剂,用于诊断疾病,纳米颗粒自身的一些性质也能杀死细胞,如光热效应等。同时,纳米材料也在电子学、光学、催化等领域有很多用途,由于纳米材料的广泛应用,人们逐渐开始关注纳米材料对人体的危害,亟需系统的评价纳米材料在体内的分布、代谢、排泄、以及累积造成的器官损伤等,现已形成一个新的研究领域,纳米毒理学。不论是纳米医学,还是纳米毒理学,都离不开了解最基本的纳米颗粒与蛋白质和细胞的相互作用,纳米材料所有的生物学效应,从蛋白质吸附,改变蛋白质构象,到特异性的细胞结合与内吞,杀死病变细胞,再到肝脾累积、氧化损伤,都是基于纳米颗粒与蛋白质和细胞的相互作用,所以有必要研究这种相互作用,一方面可以指导设计构建安全有效的纳米载体,另一方面可以控制并减少纳米材料造成的人体危害。通常,纳米材料失控的细胞毒性,如造成的细胞功能紊乱、细胞周期阻滞、甚至让细胞凋亡坏死,都是由于纳米材料与细胞的非特异性相互作用造成的,这些非特异性的作用,包括静电作用、疏水作用、氢键作用、空间效应、π键堆积等,其中静电作用是非常重要的一种作用力,目前很多研究纳米材料与细胞的静电作用仅局限于选择三种代表性的纳米材料：带正电的、中性的和带负电的纳米材料,而没有考虑表面电荷密度的影响,也没有考虑其它非特异性作用的干扰。为了研究不同表面电荷密度的纳米材料与细胞的静电作用和尽量减少其它非特异性作用的干扰,通过调节结构基本一致的带电配体和中性配体的比例,我们合成了一个表面电荷密度连续变化的金纳米颗粒阵列,这个阵列共17种纳米材料,尺寸为5nm左右的园形纳米颗粒,用元素分析法和碘切后HPLC/MS/CLND分析法定量纳米颗粒表面的各种配体的数目,依此计算出阵列的表面电荷密度,如果把100%覆盖单电荷配体的纳米颗粒的表面电荷定义为1.0,此阵列的表面电荷密度变化范围为：+2.87到-4.18。用此阵列在25和50μg/mL两种浓度下,与细胞孵育12hrs后,细胞的摄取量和表面电荷密度之间并没有线性关系,负电荷和中性的纳米颗粒几乎没有细胞摄取,在GNP05(表面电荷密度+0.52)处,有一个拐点,细胞摄取量开始大量增加,GNP01到GNP04(表面电荷密度从+287到+1.0)细胞摄取量都在最大处,没有显著性差别。可能是由于掩蔽等原因,只有暴露在最外层的表面电荷才能与细胞发生静电作用,表面正电荷密度进一步增加,也不会增加纳米颗粒的摄取量,这些细胞摄取纳米颗粒的差异,进一步用时间依赖的细胞摄取、摄取纳米颗粒细胞的TEM照片和摄取纳米颗粒细胞的暗场显微镜照片进行验证。测量纳米颗粒在水中的Zeta电位,发现在GNP09(中性纳米颗粒)处,有一个Zeta电位从正到负的转变,这种变化趋势类似于细胞摄取量的变化趋势,Zeta电位更能反映纳米颗粒在溶液中真实的静电性质。当纳米颗粒分散在含血清细胞培养基中,其表面会吸附蛋白质,形成蛋白质冠状物,SDS-PAGE分离吸附到纳米颗粒上的蛋白质,发现所有纳米颗粒的表面都结合了一定量的蛋白质,除GNP01外,其它纳米颗粒结合蛋白质的量,从SDS-PAGE上看不出明显差异,用LC/MS/MS鉴定结合到纳米颗粒上蛋白质的种类,发现纳米颗粒结合的蛋白质种类在60种左右,且有30种左右的蛋白质都能结合到所鉴定的四种纳米颗粒上。测量吸附蛋白质纳米颗粒的Zeta电位发现,吸附的蛋白质使阵列里所有纳米颗粒在溶液里的Zeta电位都是负的,且没有差异,但是吸附蛋白质纳米颗粒的细胞摄取是差异,可能吸附蛋白质在纳米颗粒表面有一个结合与解离的动态过程,并不能决定纳米颗粒与细胞的静电作用。我们发现,纳米颗粒的表面电荷密度,纳米颗粒的Zeta电位,和吸附蛋白质纳米颗粒的Zeta电位,这三种描述纳米材料带电性质的参数,都不符合纳米材料带电性质与细胞摄取的关系,如果把纳米颗粒最外层的表面电荷密度定义为有效表血电荷密度,有效表面电荷密度可以解释正电荷的密度过高后,细胞摄取量会不再增加,所以说有效表面电荷密度决定了纳米颗粒与细胞的静电作用。同时,虽然正电荷纳米颗粒能大量被细胞摄取,前提条件是其表面的正电荷密度需要达到一定量,以提供足够大的静电作用力,引发细胞的内吞行为。蛋白质吸附能让正电荷纳米颗粒的表面电性变成负的,但纳米颗粒表面的正电荷基团仍能暴露出来并吸附到细胞表面。纳米颗粒用于载药时,为了增强特异性,提高药效,通常会在纳米颗粒表面修饰抗体或其它靶向分子,这些靶向分子能与病变细胞表面特有的受体或过表达的受体特异性的结合,从而只进入并杀死病变组织或细胞,不会产生副作用。但是,当纳米颗粒进入到血液等生理环境中,其表面会吸附蛋白质等生物大分子,形成蛋白质冠状物,这种蛋白质冠状物会影响纳米颗粒与细胞的相互作用。目前,关于这种蛋白质吸附是否会影响纳米颗粒靶向性的研究仍很少,人们在设计靶向性纳米颗粒时,也很少会考虑蛋白质吸附的影响,为了研究吸附蛋白质对纳米颗粒靶向性的影响,我们合成了三种尺寸的靶向性金纳米颗粒,根据纳米颗粒的TEM照片,统计的粒径分别是GNP-5:45±2.5nm,GNP-15:14.2±1.6nm,GNP-40:38.8±49nm。SDS-PAGE显示纳米颗粒分散在含血清的细胞培养基中,其表面将吸附蛋白质。吸附蛋白质的靶向性纳米颗粒和靶向性纳米颗粒自身相比,细胞摄取量有差异,这些差异与纳米颗粒的尺寸、剂量以及靶向受体的密度有关。小粒径的纳米颗粒,曲率大,靶向分子与细胞表面受体间不易发生多键作用,吸附的蛋白质易阻碍靶向分子与受体的结合,从而使其摄取量下降较多。大粒径的纳米颗粒,曲率小,由于靶向分子与细胞表面受体间存在多键作用,吸附的蛋白质不能完全阻碍靶向分子与受体的结合,同时吸附的蛋白质也会与细胞表面受体间存在多键作用,提供了额外的作用力,使大粒径纳米颗粒的细胞摄取量略微增加。中等尺寸的纳米颗粒,在高剂量时,表现为摄取的阻碍,在低剂量时,表现为摄取的增加,可能是由于中等尺寸纳米颗粒的靶向分子与细胞表面受体间存在多种作用方式,低剂量时,能与靶向分子结合的受体没有饱和,会发生多键作用；高剂量时,受体结合达到饱和,发生多键作用的概率降低。降低可结合靶向受体的密度后,小粒径纳米颗粒的细胞摄取量,在蛋白质吸附前后,差异变小甚至无差异,这是因为此时,所有纳米颗粒都不易与受体结合,都不易被细胞摄取,从而让摄取量差异变小。而结合蛋白质的大粒径纳米颗粒的细胞摄取量始终多于纳米颗粒自身,因为大粒径纳米颗粒结合的蛋白质能与细胞表面发生多种蛋白质结合的多键作用,从而增加其细胞摄取量。基于以上研究,我们认为吸附蛋白质对纳米材料的靶向性是有影响的,因此在设计靶向纳米载体时,不仅需要考虑载体的尺寸,而且要考虑临床所用剂量和靶向受体的密度。
[Abstract]:Nano materials have potential medical value, can be used for drug loading, loading gene, as therapeutic agents, can also be used as a contrast agent for the diagnosis of diseases, some properties of the nanoparticles themselves can kill cells, such as photothermal effect. At the same time, nano materials in electronics, optics, catalysis and other fields have many uses, because application of nano materials, people gradually began to pay attention to the harm of nano materials on the human body, to the evaluation system of nano materials in vivo distribution, metabolism, excretion, and the cumulative result of organ damage, has become a new research field, nano toxicology. Whether nano medicine, or nano toxicology, are inseparable from the open the most basic understanding of the interaction of nanoparticles and proteins and cells, the biological effects of nanomaterials from all the protein adsorption, changes in protein conformation, to specific cell binding With endocytosis, kill diseased cells, and then to the liver and spleen accumulation, oxidative damage, is the interaction of nanoparticles and proteins and cell based, so it is necessary to study this interaction, one can guide the design of safe and effective construction of nano carrier, on the other hand can control and reduce the harm to human body caused by nano materials.
Usually, the cytotoxicity of nano materials is out of control, such as the cause of the disorder of cell function, cell cycle arrest, apoptosis and even necrosis, are the result of nonspecific nanomaterials and cell interaction, these nonspecific effects, including electrostatic interaction, hydrophobic interaction, hydrogen bonding, space effect, pi bond accumulation so, the electrostatic interaction is a very important force, the electrostatic interaction research of nano materials and many cells are only limited to choose three representative nanomaterials: positive, neutral and negatively charged nano materials, without considering the effect of surface charge density, did not consider other nonspecific interference the role of electrostatic interaction. In order to study different nano materials and cell surface charge density and reduce the interference of other nonspecific effects, by adjusting the structure consistent with Electric ligands and neutral ligands, we synthesized Au nanoparticles with a continuous change of the surface charge density of the array, a total of 17 kinds of nano materials, the size of circular nano particles of about 5nm, the number of analysis method for quantitative analysis of various ligand surface nano particles HPLC/MS/CLND and iodine after digestion by element, so calculated the surface charge density of the array, if the surface charge defined nanoparticles covering 100% single charge ligands is 1, the surface charge density range of this array is +2.87 to -4.18. with the array at 25 and 50 g/mL two concentrations, incubation of cells with 12hrs, and there is no linear relationship between cells the uptake of nanoparticles and surface charge density, negatively charged and neutral almost no cell uptake in GNP05 (surface charge density +0.52), there is a turning point, cell intake began to increase, GNP0 1 to GNP04 (surface charge density from +287 to +1.0) cell intake are among the most, there is no remarkable difference. May be due to masking and other reasons, only the exposure occurred in the surface charge and electrostatic effect to cells in the outer layer, surface positive charge density further increased, will not increase the intake of nanoparticles these differences in cellular uptake of nanoparticles and cell uptake further with time dependent, dark field microscope photos of the TEM photos and the uptake of nanoparticles uptake nanoparticles cells were used. To measure the particle Zeta potential in water, found in GNP09 (neutral nanoparticles), there is a change from positive to negative Zeta potential the change trend, this trend is similar to the cellular uptake of electrostatic properties of Zeta can better reflect the true potential of nano particles in the solution. When the nano particles dispersed in the serum containing cell culture Medium will protein adsorption on its surface, the formation of protein coronary, SDS-PAGE dissociation in the nanoparticle protein, found all surfaces of nano particles with a certain amount of protein, in addition to GNP01, the amount of protein with other nanoparticles, from SDS-PAGE showed no obvious difference, identified by LC/MS/MS binding to the the type of protein nanoparticles, nanoparticles combined with protein species in the 60 or so, and there are four kinds of nanoparticles about 30 kinds of protein can bind to the identification. Zeta potential measurement of protein adsorption of nanoparticles, the adsorption of proteins in all the array of nanoparticles in solution of Zeta potential are all negative, and there is no difference, but the cellular uptake of nanoparticles is differences in protein adsorption, the dynamic adsorption may have a protein binding and dissociation in the particle surface. The process, does not determine the electrostatic interaction between nanoparticles and cells. We found that the surface charge density of nano particles, the Zeta potential of nanoparticles, and the adsorption of protein nanoparticles Zeta potential, the three charged description of the properties of nano materials parameters do not meet the relationship between nano materials and electrical properties of cellular uptake, if the surface charge density of the definition of the outermost layer of nano particles as the effective surface charge density of the blood, the effective surface charge density can explain the positive charge density is too high, the cell uptake will no longer increase, so that the surface electric charge density effectively determines the electrostatic interaction between nanoparticles and cells. At the same time, although the positively charged nanoparticles can be a large number of cells intake condition is the positive charge density of the surface to achieve a certain amount, in order to provide electrostatic force is large enough, triggering endocytosis behaviors of cells. The protein adsorption The surface electricity of the positive charge nanoparticles can be turned negative, but the positive charge group on the surface of the nanoparticles can still be exposed and adsorbed on the surface of the cell.
Nanoparticles for drug loading, in order to enhance the specificity, improve efficacy, usually to the molecules in the surface of nano particle modified antibodies or other targets, the target molecule can bind to cell surface receptors or disease specific overexpression of the receptor specific, only to enter and kill diseased tissues or cells, can not produce side effects. However, when the nano particles into the blood physiological environment, the surface adsorption of proteins and other biological macromolecules, the formation of protein coronary, the protein corona will affect the interactions of nanoparticles and cells. At present, the protein adsorption will affect the nanoparticle targeted research is still very few, people in the design of targeted nanoparticles, rarely consider the impact of protein adsorption on nanoparticles, in order to study the effect of targeted protein adsorption, we synthesized three kinds of dimensions The targeting of gold nanoparticles, nano particles of TEM according to the photos, statistics of particle size are GNP-5:45 + 2.5nm, GNP-15:14.2 + 1.6nm, GNP-40:38.8 + 49nm.SDS-PAGE showed that nano particles dispersed in serum-free cell culture medium, the surface adsorption of protein. The protein adsorption of targeted nanoparticles and targeting nano the particles themselves compared to cell uptake differences, these differences and the particle size, dosage and targeted receptor density. Nanoparticles with small particle size, large curvature, target molecules and cell surface receptors is not easy to be multi key role, combined with the protein adsorption to hinder targeting molecules and receptors thus, the intake decreased more. Nanoparticles, large diameter curvature is small, due to targeted molecular and cell surface receptors between multi key role, the adsorbed protein can not impede the targeting molecule and receptor The combination of the simultaneous adsorption of proteins also exist multiple bond interaction with cell surface receptors, provides additional force, cell intake slightly to large particle size of nanoparticles increased. Nano particles of medium size, at high doses, showed uptake of obstacles, at low doses, showed uptake increase may be due to medium size nanoparticles targeting molecules and cell surface receptors exist between the various modes of action, low doses, can be combined with targeted molecular receptors is not saturated, will happen more key effect; high doses, receptor binding is saturated, reduce the probability of occurrence of multiple key role. Decreased combined with the targeted receptor density, cell uptake of small size nanoparticles, protein adsorption before and after, the difference became smaller or even no difference, this is because at this time, all particles are not easy and receptor binding, are not easy to be cell So the intake, intake difference becomes smaller. And combined with the cellular uptake of large amount of grain protein particles is always more than the nanoparticles themselves, because of the large size of nano particle binding protein can occur multiple bond interaction with various proteins and the cell surface, thereby increasing the cell uptake. Based on the above research, we think protein adsorption on nano materials targeting is influential, so in the design of targeted nano carrier, not only need to consider the size of the carrier, but also to consider the clinical dose and targeting receptor density.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：TB383.1;R318.08

【共引文献】