用于内毒素去除的新型亲和吸附剂的制备及性能研究

【作者】 张媛媛；

【导师】 平郑骅；

【作者基本信息】 复旦大学 ， 高分子化学与物理， 2007， 博士

【摘要】 随着生物制药和生物医学工程相关领域的发展,人们对产品中可能存在的微量有害物质的清除越来越重视。内毒素（ET）是一种在自然界广泛存在的污染物,是一种毒性很强的致炎和热原物质。有效清除制品中、特别是生物制品中污染的内毒素一直是医学、制药、生物工程等领域研究的热点。由于内毒素的相对分子质量较大,与大分子生物制品在同一数量级,因此从这类制品中去除微量的内毒素难度很大。迄今为止,在实际生产过程中得到应用的材料和方法并不多。一些已经商品化的产品也存在吸附效率低,价格高等问题。因此,对这一领域的研究仍具有十分重要的价值和实际意义。本课题针对当前内毒素亲和吸附剂存在的问题,从载体、配基两方面着手,结合反应条件的研究,发展了几种新型的适于用作ET吸附的活性配基物质,制备出了对内毒素吸附量高、特异性好、血液相容性好的新型吸附材料。通过对这些材料的吸附特性的研究,为内毒素亲和吸附剂的进一步结构设计和制备提供依据。在载体材料的研究方面,论文主要研究了作为活性吸附剂载体的多孔硅胶的孔径和比表面等参数对亲和吸附剂制备的影响。以多孔硅胶为载体的亲和吸附剂是采用表面接枝的方法制备的。选用组氨酸（His）为活性配基。经酸化处理的多孔硅胶先以硅烷偶联剂KH550硅烷化,在硅胶表面引入活性胺基,然后以戊二醛为间隔臂将组氨酸接枝在多孔硅胶载体上,制成用于内毒素去除的亲和吸附剂。用IR,TGA、元素分析和功能基化学滴定的方法测定了每步反应的转化率,即不同反应物在多孔硅胶上的接枝率。结果表明,反应物在硅胶上的含量与硅胶的比表面积及孔径有关,且多步反应使硅胶载体的孔径不断减小。由于ET的分子尺寸较大,因此,选用较大孔径的多孔硅胶基质制成的亲和吸附剂对ET的吸附效果较好。这样才能使ET分子扩散到硅胶孔内被接枝在孔壁上的活性配基吸附。在配基的研究方面,本论文研究了三种不同的配基:一种是文献上已有大量报道的组氨酸配基,一种是根据组氨酸中咪唑基对ET的特殊亲和作用而选则的乙烯基咪唑配基（VI）,最后一种是在实验过程中发现的含环氧基的亲和配基。前两种配基中的主要活性位均是咪唑基团,但组氨酸分子中还含有负电性的羧基和具反应活性的胺基。乙烯基咪唑是通过自由基聚合反应接枝到硅胶载体上的,得到的是一种大分子配基。通过上述两种吸附剂对水溶液中ET的静态和动态吸附性能进行比较,发现以聚乙烯咪唑为配基的吸附剂对ET有更高的吸附容量和去除率。这不仅证明了咪唑基作为活性功能基对ET有很高的选择吸附性,而且也证明,配基与ET之间的静电相互作用是使其具有较好吸附特性的主要原因。当配基分子中含有羧基等负电性基团时,会削弱咪唑基对ET的吸附作用。含环氧基的化合物作为ET的亲和配基至今未见文献报道。这种含环氧基的配基可以通过与相应的硅烷偶联剂反应引入吸附剂表面,或是通过在含端烯基的载体上接枝甲基丙烯酸缩水甘油酯（GMA）而固载到载体上。研究表明,一旦环氧基被打开,相应的吸附剂对ET就不再呈示吸附能力,说明环氧基团是对ET起选择吸附作用的活性位。有关该配基与ET作用的机理还有待于进一步的深入研究。但这一发现拓宽了活性配基选择的范围。聚乙烯基咪唑作为大分子配基用于ET的吸咐虽然有很高的选择性和吸附容量,但VI较强的分子间作用力及咪唑基过高的接枝密度会显著影响该配基的吸附效率。论文通过VI与甲基丙烯酸酯的共聚较好地调节了咪唑基在接枝链上的分布。由于VI与甲基丙烯酸酯在溶液中发生共聚反应的竞聚率有很大差别,属r1<1,r2>1的非恒比共聚反应。所以在共聚时,乙烯基咪唑自聚的能力会很差。因此,在与甲基丙烯酸酯单体形成的共聚链中绝大多数乙烯基咪唑单元会被若干个丙烯酯单元隔开成单个分布。可以认为,对ET基本无吸附效果的丙烯酸酯在共聚体中发挥了间隔臂的作用,使单位咪唑基对ET的吸附量得到提高。论文通过共聚接枝的方法还有反相悬浮聚合的方法制备了一系列VI与第二单体的共聚粒子,并测定了不同单体配比下得到的共聚接枝物对ET的吸附性能。在合成路线方面,除了KH550方法外,论文还研究了用环氧法引入组氨酸配基的方法。首先用含环氧基的偶联剂KH560对硅胶载体进行硅烷化处理,引入环氧基,然后利用环氧基与组氨酸分子上的氨基直接开环加成,或利用将环氧基氧化形成的醛基为中介进行加成,使组氨酸配基结合在硅胶载体上。结果表明,直接开环加成制成的吸附剂对ET有更高的吸附容量。论文对吸附剂的各种制备方法中配基接枝反应的条件进行了详细研究。如温度、时间、介质的pH、单体浓度等。例如,在作为间隔臂和桥连剂的戊二醛与硅胶上端胺基的反应中,温度和介质的pH有很大影响。当反应温度低于50℃,介质的pH为中性条件时,二者加成所得产物中的活性醛基相对含量较高。而在高温和碱性环境下反应时,戊二醛会发生自聚,虽然用TGA测得的戊二醛在硅胶上的含量较高,但活性醛基的含量却很低。因此,反应条件的控制十分重要。论文研究了不同方法制备的亲和吸附剂对ET的吸附特性。发现以硅胶为载体,组氨酸为配基的亲和吸附剂在水中对ET的等温吸附符合典型的Langmuir模型,吸附过程约2h达平衡。其饱和吸附量q_m和表观解离常数K_d分别为1194μg/g和1.35×10^-9M。该吸附剂在血清或蛋白溶液中对ET的去除率大于85%,且对血清中各成份的非选择性吸附很小,具有良好的血液相容性。PVI-接枝得到的吸附剂对ET吸附80min可达平衡,吸附动力学符合二级方程,且速率常数k₂随起始ET浓度的增高而降低。ET在PVI/Silica gel粒子表面的等温吸附较好地满足Freundlich方程。测得常数K_f为109.6,n=1.35。在更高的ET初始浓度下,该吸附剂的等温线行为呈现bimodal型,与文献报道的以聚合物为配基的吸附剂的典型等温线相似。吸附剂的平衡吸附率随温度升高略增。在中性条件下,PVI-silica gel吸附剂对BSA蛋白溶液中的ET去除率大于96%。本论文的研究表明,硅胶是一种适用于制备ET亲和吸附剂的优良载体。以乙烯基咪唑为活性配基,以硅胶为载体制成的亲和吸附剂对ET具有良好的吸附选择性和去除能力,且较少受介质的pH和离子强度的影响。制备简便,性能稳定,是一种具有良好应用前景的亲和吸附剂。更多还原

【Abstract】 Now with the research development in field of biomedicine and bioengineering, more and more importance are attached to the clearance of trace hazardous substances possibly exist in the product. Endotoxin is a kind of potential contaminant broadly existing everywhere and may cause serious pyrogenic and shock reactions. So the removal of endotoxin from system especially from the bio-preparations is becoming increasingly crucial and has remained an important research aim. Among these the ET removal from macromolecular substance-containing solutions is rather difficult. Till now only a little method and material has gained practical application which, however, suffers from low removal efficiency and high expense. So the study in this field is still valuable and significative.The thesis of this dissertation comes from a Med-X fund cooperative program of Fudan University and Zhongshan Hospital. According to the problems of the present ET affinity adsorbent, some novel affinity adsorbents for ET removal were developed in this dissertation starting with matrix and ligand, which were proven to have high adsorption capacity, selectivity and compatibility to blood. And the study on preparation and ET adsorption properties of these adsorbents could provide reference for the further design of ET removal materials.As for the matrix, the porous silica gel was mainly selected and the influence of its pore size and specific surface area on the property of the resultant adsorbent was studied. His was used as ligand here, and was grafted on the KH550 modified silica gel through the link with glutaraldehyde (Glu). The grafting yield of the functional group in each step were studied by means of IR, thermogravimetric analysis (TGA), elemental analysis and chemical titration method. The results show that the grafting yield of each reactant on silica gel is affected by the pore size and specific surface area of the silica gel matrix, and during the successional reactions the pore size continually decreases. Since ET has a large molecule, it is better to choose the matrix with a larger pore size to allow the diffusion of ET into the internal pore walls and to be catched by the ligand on it.As for the ligand, three ligands were investigated in this dissertation. They are: Histidine (His), which has been largely reported in literature; vinylimidazole (VI), selected here from the elicitation of the affinity mechanism of imidazole group in His; and the epoxy group-containing compound, discovered during our experiment. The active site of His and VI are both imidazole group, but His also has reactive amido and -COOH with negative charge. The ligand obtained from VI graft polymerization on silica gel is a kind of polymeric form. Through comparison between the ET removal performance of the affinity adsorbent with His and PVI as ligand respectively, a much higher capacity, efficiency and much better applicability in terms of pH and ionic strength of PVI ligand was found, which proves not only the high affinity of the active imidazole group toward ET, but also the leading function of ionic interaction between ligand and ET molecule. The epoxy-containing compound such as KH560 or GMA for use as ET ligand has never been reported yet. It was found that once the epoxy group was opened, the resultant adsorbent couldn’t show ET removal ability any longer, which verifies the function of epoxy group. This finding extends the scope of ET ligand selection.Although PVI as a polymeric ligand showed high ET removal capacity, it was still affected by the too high graft density and the strong forces between its molecules. By the copolymerization of VI and methacrylate monomers, the distribution of VI chains on the matrix could be adjusted and improved. Since the reactivity ratios of VI and the methacrylates are widely discrepant, almost each VI unit in the copolymer chain will be isolated by the methacrylate segment, thus the ET removal capacity of per VI unit was increased as a result of the presence of polymeric spacer arm.As for the graft route of ligand, besides KH550, another method using KH560 for the activation of silica gel to introduce His ligand on it was studied. Two kinds of reactions were then conducted to introduce His on the epoxy-silica gel. The ET removal tests show that the direct opening of epoxy group to react with His is superior to the oxidation-addition method which use -CHO as the media reactive group.In the preparation process of these affinity adsorbents, the reaction conditions such as temperature, time, pH, concentration of reactant for His introduction on KH550 or KH560 modified silica gel and for PVI graft polymerization on KH570 modified silica gel were studied in detail. For example, the reaction of Glu with amido-silica gel was found to be greatly influenced by temperature and pH value of the reaction medium. At temperature lower than 50℃and in neutral pH, the highest content of active -CHO could be obtained. At higher temperatures than 50℃and more basic conditions, self-polymerization of Glu will occur, and the content of -CHO decreased despite the high grafting yield of Glu by TGA. Again, in the graft reaction of PVI, a series of adsorbents with PVI content (grafting degree) from 1.1% to 16% could be obtained by changing the conditions, which showed different ET removal efficiency. So the control of reaction conditions is important.The ET removal properties of the above affinity adsorbents prepared by different method or with different ligands were studied. The adsorption isotherm of His-Glu-KH550-silica gel adsorbent showed good accordance with the Langmuir model, the maximum adsorption capacity q_m and apparent dissociation constant K_d were calculated to be 1194 g/g and 1350 g/L. The adsorbent had little nonspecific adsorption of protein and other components in a serum-endotoxin solution, with a ET removal efficiency of above 85%. The isotherms of PVI-silica gel displayed well fit for Freundlich isotherm model, which is indicative of surface heterogeneity of the sorbent. The Freundlich constants K_f and n were calculated to be 109.6 EU/mg and 1.35. The adsorption kinetics was well accorded with the second order equation. The equilibrium ET adsorption efficiency increased slightly with the temperature.The study of this dissertation reveals that silica gel is a good matrix for ET affinity adsorption preparation, and PVI-silica gel adsorbent is promising in ET removal.更多还原