本文選題：金納米顆粒 + 表面電荷密度　； 參考：《山東大學》2013年博士論文

【摘要】：納米材料具有潛在醫(yī)學價值,可以用于載藥、載基因等,作為治療試劑,也可以作為造影劑,用于診斷疾病,納米顆粒自身的一些性質(zhì)也能殺死細胞,如光熱效應(yīng)等。同時,納米材料也在電子學、光學、催化等領(lǐng)域有很多用途,由于納米材料的廣泛應(yīng)用,人們逐漸開始關(guān)注納米材料對人體的危害,亟需系統(tǒng)的評價納米材料在體內(nèi)的分布、代謝、排泄、以及累積造成的器官損傷等,現(xiàn)已形成一個新的研究領(lǐng)域,納米毒理學。不論是納米醫(yī)學,還是納米毒理學,都離不開了解最基本的納米顆粒與蛋白質(zhì)和細胞的相互作用,納米材料所有的生物學效應(yīng),從蛋白質(zhì)吸附,改變蛋白質(zhì)構(gòu)象,到特異性的細胞結(jié)合與內(nèi)吞,殺死病變細胞,再到肝脾累積、氧化損傷,都是基于納米顆粒與蛋白質(zhì)和細胞的相互作用,所以有必要研究這種相互作用,一方面可以指導設(shè)計構(gòu)建安全有效的納米載體,另一方面可以控制并減少納米材料造成的人體危害。 通常,納米材料失控的細胞毒性,如造成的細胞功能紊亂、細胞周期阻滯、甚至讓細胞凋亡壞死,都是由于納米材料與細胞的非特異性相互作用造成的,這些非特異性的作用,包括靜電作用、疏水作用、氫鍵作用、空間效應(yīng)、π鍵堆積等,其中靜電作用是非常重要的一種作用力,目前很多研究納米材料與細胞的靜電作用僅局限于選擇三種代表性的納米材料：帶正電的、中性的和帶負電的納米材料,而沒有考慮表面電荷密度的影響,也沒有考慮其它非特異性作用的干擾。為了研究不同表面電荷密度的納米材料與細胞的靜電作用和盡量減少其它非特異性作用的干擾,通過調(diào)節(jié)結(jié)構(gòu)基本一致的帶電配體和中性配體的比例,我們合成了一個表面電荷密度連續(xù)變化的金納米顆粒陣列,這個陣列共17種納米材料,尺寸為5nm左右的園形納米顆粒,用元素分析法和碘切后HPLC/MS/CLND分析法定量納米顆粒表面的各種配體的數(shù)目,依此計算出陣列的表面電荷密度,如果把100%覆蓋單電荷配體的納米顆粒的表面電荷定義為1.0,此陣列的表面電荷密度變化范圍為：+2.87到-4.18。用此陣列在25和50μg/mL兩種濃度下,與細胞孵育12hrs后,細胞的攝取量和表面電荷密度之間并沒有線性關(guān)系,負電荷和中性的納米顆粒幾乎沒有細胞攝取,在GNP05(表面電荷密度+0.52)處,有一個拐點,細胞攝取量開始大量增加,GNP01到GNP04(表面電荷密度從+287到+1.0)細胞攝取量都在最大處,沒有顯著性差別�？赡苁怯捎谘诒蔚仍�,只有暴露在最外層的表面電荷才能與細胞發(fā)生靜電作用,表面正電荷密度進一步增加,也不會增加納米顆粒的攝取量,這些細胞攝取納米顆粒的差異,進一步用時間依賴的細胞攝取、攝取納米顆粒細胞的TEM照片和攝取納米顆粒細胞的暗場顯微鏡照片進行驗證。測量納米顆粒在水中的Zeta電位,發(fā)現(xiàn)在GNP09(中性納米顆粒)處,有一個Zeta電位從正到負的轉(zhuǎn)變,這種變化趨勢類似于細胞攝取量的變化趨勢,Zeta電位更能反映納米顆粒在溶液中真實的靜電性質(zhì)。當納米顆粒分散在含血清細胞培養(yǎng)基中,其表面會吸附蛋白質(zhì),形成蛋白質(zhì)冠狀物,SDS-PAGE分離吸附到納米顆粒上的蛋白質(zhì),發(fā)現(xiàn)所有納米顆粒的表面都結(jié)合了一定量的蛋白質(zhì),除GNP01外,其它納米顆粒結(jié)合蛋白質(zhì)的量,從SDS-PAGE上看不出明顯差異,用LC/MS/MS鑒定結(jié)合到納米顆粒上蛋白質(zhì)的種類,發(fā)現(xiàn)納米顆粒結(jié)合的蛋白質(zhì)種類在60種左右,且有30種左右的蛋白質(zhì)都能結(jié)合到所鑒定的四種納米顆粒上。測量吸附蛋白質(zhì)納米顆粒的Zeta電位發(fā)現(xiàn),吸附的蛋白質(zhì)使陣列里所有納米顆粒在溶液里的Zeta電位都是負的,且沒有差異,但是吸附蛋白質(zhì)納米顆粒的細胞攝取是差異,可能吸附蛋白質(zhì)在納米顆粒表面有一個結(jié)合與解離的動態(tài)過程,并不能決定納米顆粒與細胞的靜電作用。我們發(fā)現(xiàn),納米顆粒的表面電荷密度,納米顆粒的Zeta電位,和吸附蛋白質(zhì)納米顆粒的Zeta電位,這三種描述納米材料帶電性質(zhì)的參數(shù),都不符合納米材料帶電性質(zhì)與細胞攝取的關(guān)系,如果把納米顆粒最外層的表面電荷密度定義為有效表血電荷密度,有效表面電荷密度可以解釋正電荷的密度過高后,細胞攝取量會不再增加,所以說有效表面電荷密度決定了納米顆粒與細胞的靜電作用。同時,雖然正電荷納米顆粒能大量被細胞攝取,前提條件是其表面的正電荷密度需要達到一定量,以提供足夠大的靜電作用力,引發(fā)細胞的內(nèi)吞行為。蛋白質(zhì)吸附能讓正電荷納米顆粒的表面電性變成負的,但納米顆粒表面的正電荷基團仍能暴露出來并吸附到細胞表面。 納米顆粒用于載藥時,為了增強特異性,提高藥效,通常會在納米顆粒表面修飾抗體或其它靶向分子,這些靶向分子能與病變細胞表面特有的受體或過表達的受體特異性的結(jié)合,從而只進入并殺死病變組織或細胞,不會產(chǎn)生副作用。但是,當納米顆粒進入到血液等生理環(huán)境中,其表面會吸附蛋白質(zhì)等生物大分子,形成蛋白質(zhì)冠狀物,這種蛋白質(zhì)冠狀物會影響納米顆粒與細胞的相互作用。目前,關(guān)于這種蛋白質(zhì)吸附是否會影響納米顆粒靶向性的研究仍很少,人們在設(shè)計靶向性納米顆粒時,也很少會考慮蛋白質(zhì)吸附的影響,為了研究吸附蛋白質(zhì)對納米顆粒靶向性的影響,我們合成了三種尺寸的靶向性金納米顆粒,根據(jù)納米顆粒的TEM照片,統(tǒng)計的粒徑分別是GNP-5:45±2.5nm,GNP-15:14.2±1.6nm,GNP-40:38.8±49nm。SDS-PAGE顯示納米顆粒分散在含血清的細胞培養(yǎng)基中,其表面將吸附蛋白質(zhì)。吸附蛋白質(zhì)的靶向性納米顆粒和靶向性納米顆粒自身相比,細胞攝取量有差異,這些差異與納米顆粒的尺寸、劑量以及靶向受體的密度有關(guān)。小粒徑的納米顆粒,曲率大,靶向分子與細胞表面受體間不易發(fā)生多鍵作用,吸附的蛋白質(zhì)易阻礙靶向分子與受體的結(jié)合,從而使其攝取量下降較多。大粒徑的納米顆粒,曲率小,由于靶向分子與細胞表面受體間存在多鍵作用,吸附的蛋白質(zhì)不能完全阻礙靶向分子與受體的結(jié)合,同時吸附的蛋白質(zhì)也會與細胞表面受體間存在多鍵作用,提供了額外的作用力,使大粒徑納米顆粒的細胞攝取量略微增加。中等尺寸的納米顆粒,在高劑量時,表現(xiàn)為攝取的阻礙,在低劑量時,表現(xiàn)為攝取的增加,可能是由于中等尺寸納米顆粒的靶向分子與細胞表面受體間存在多種作用方式,低劑量時,能與靶向分子結(jié)合的受體沒有飽和,會發(fā)生多鍵作用；高劑量時,受體結(jié)合達到飽和,發(fā)生多鍵作用的概率降低。降低可結(jié)合靶向受體的密度后,小粒徑納米顆粒的細胞攝取量,在蛋白質(zhì)吸附前后,差異變小甚至無差異,這是因為此時,所有納米顆粒都不易與受體結(jié)合,都不易被細胞攝取,從而讓攝取量差異變小。而結(jié)合蛋白質(zhì)的大粒徑納米顆粒的細胞攝取量始終多于納米顆粒自身,因為大粒徑納米顆粒結(jié)合的蛋白質(zhì)能與細胞表面發(fā)生多種蛋白質(zhì)結(jié)合的多鍵作用,從而增加其細胞攝取量�；谝陨涎芯�,我們認為吸附蛋白質(zhì)對納米材料的靶向性是有影響的,因此在設(shè)計靶向納米載體時,不僅需要考慮載體的尺寸,而且要考慮臨床所用劑量和靶向受體的密度。
[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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：TB383.1;R318.08

【共引文獻】

相關(guān)期刊論文 前10條

1 文明;柏瑋;李必波;韓忠;陸云峰;李少林;楊學恒;;SPIO標記的反義探針磁學性能檢測[J];重慶大學學報;2008年08期

2 柴學;張帆;盧光明;;氧化鐵顆粒在MR分子影像研究中的進展[J];放射學實踐;2009年05期

3 張景峰;張敏鳴;;腫瘤血管生成的影像學研究及進展[J];國外醫(yī)學(臨床放射學分冊);2007年06期

4 陳銦銦;郭亮;;USPIO在評價腫瘤微血管生成中的作用及其臨床應(yīng)用[J];國際醫(yī)學放射學雜志;2009年04期

5 容蓉;丁杰;王霄英;姜原;蔡文超;邱建星;宋玉軍;;雙能CT應(yīng)用于分子影像學的可行性初探——模體實驗[J];放射學實踐;2012年07期

6 葛小林;孟慶紅;馬s，

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