【作者】 潘晓赟；

【导师】 邵正中； 姚萍；

【作者基本信息】 复旦大学 ， 高分子化学与物理， 2008， 博士

【摘要】 由两亲性共聚物组成的胶束已经被开发用作水相体系中的疏水药物载体。用合成高分子作为构建药物载体的材料一直是研究的重点。相比合成高分子而言,天然高分子具有许多独特的性质,它们既可以包埋疏水药物,又可以包埋亲水药物,所以天然高分子也可以是构建药物载体的好材料。更值得一提的是,天然高分子有较高的安全性。用天然高分子通过简便易行的绿色化学方法构建药物载体,可以有效地避免合成化学试剂和有机溶剂的使用,这对载体在生物医药工业中的应用非常有利。酪蛋白(casein)是牛奶中的主要组成成分,它是一组蛋白的磷酸盐,由四种成分组成:αs1-,αs2-,β-和κ-酪蛋白,这四种成分在牛奶中的质量比约为3:0.8:3:1,它们的分子量在19000-25000Da范围内。这四种酪蛋白都是两亲性的蛋白质,没有确定的结构。在食品工业中,酪蛋白在多个方面具有应用价值,比如乳化,与水的结合,与油脂的结合,以及质构等。这些优点使得酪蛋白可以作为理想的基材来构造纳米载体,用于药物输送系统。Maillard反应是一种天然无毒的反应,它在食品的运输、烹饪和储存过程中都有可能发生。Maillard反应的化学原理已被广泛地研究过,今天我们已经知道这个反应的本质是通过多糖的还原性端羟基与蛋白的氨基(包括端氨基和赖氨酸的氨基)之间的反应将蛋白和多糖连接在一起。Maillard反应产物在许多领域都有广泛的应用,但用于药物包埋的却很少。本论文的工作主要集中在制备基于酪蛋白的纳米粒子并对其应用性进行研究。我们主要制备了两种纳米粒子。一种是通过Maillard反应制备酪蛋白-葡聚糖接枝共聚物,并通过调节pH值使共聚物自组装形成胶束粒子。该粒子具有疏水性内核,我们用β-胡萝卜素作为模型化合物研究了该粒子对疏水化合物的包埋以及释放性质。另外,由于Maillard反应提高了β-酪蛋白在酸性pH范围内的溶解性,我们也研究了β-酪蛋白-葡聚糖接枝共聚物作为酸性体系乳化剂的性能。另一种粒子是利用球状蛋白的凝胶化性质制备的β-酪蛋白-葡聚糖/溶菌酶纳米粒子。该粒子核的亲水性较强,我们用芘的电荷衍生物以及氯金酸作为模型化合物研究了该粒子对极性化合物的包埋行为。论文中所有的纳米粒子的制备都只使用了酸和碱以及乙醇,没有其它化学试剂和溶剂,是一种绿色化学的方法。具体来说,本论文包括了以下五个部分的工作:第一部分是Maillard反应制备酪蛋白-葡聚糖接枝共聚物及其胶束化行为的研究。我们利用Maillard反应中的Amadori重排制备了两种天然大分子-酪蛋白和葡聚糖的接枝共聚物。所制备的酪蛋白-葡聚糖接枝共聚物具有pH敏感的可逆性质:在pH值接近酪蛋白等电点时形成以酪蛋白为核,葡聚糖为壳的胶束;在pH值偏离酪蛋白等电点时,胶束会解离。共聚物形成胶束的驱动力是酪蛋白在等电点时由于净电荷为零发生聚集,而共价接枝的葡聚糖阻止了宏观沉淀的形成导致纳米粒子的生成。酪蛋白-葡聚糖接枝共聚物的胶束具有球状外貌,胶束的尺寸取决于共聚物的接枝度,以及所用葡聚糖的分子量等。该胶束具有较强的疏水性,可以包埋芘这样的疏水化合物。第二部分是Maillard反应制备β-酪蛋白-葡聚糖接枝共聚物及其乳化性质的研究。我们利用Maillard反应中的Amadori重排反应制备了β-酪蛋白-葡聚糖接枝共聚物。当介质pH值处于4.0-5.0范围内,即接近β-酪蛋白的等电点时,β-酪蛋白溶解度降低,β-酪蛋白-葡聚糖接枝共聚物可形成胶束。原子力显微镜显示该胶束具有球形外貌。共聚物的接枝度和葡聚糖侧链的长度共同决定了共聚物胶束的尺寸及其可稳定存在的时间。高接枝度的共聚物亲水性较强,以单分子状态存在于溶液中,在pH4.6和8.0都显示出较强的乳化能力。第三部分是对酪蛋白-葡聚糖接枝共聚物和β-胡萝卜素在疏水力诱导下的协同组装行为研究。酪蛋白-葡聚糖接枝共聚物在pH7.0的水溶液中以单分子状态存在,而β-胡萝卜素难溶于水。我们通过透析法或者蒸发法,将共聚物/β-胡萝卜素混合液的水/乙醇混合溶剂替换成纯水。在此过程中,酪蛋白和β-胡萝卜素通过疏水相互作用形成的复合物的溶解度降低,同时葡聚糖的溶解度上升,最终形成以酪蛋白和β-胡萝卜素复合物为核,葡聚糖为壳的纳米粒子。在纳米粒子的自组装过程中,酪蛋白-葡聚糖接枝共聚物也同时实现了对β-胡萝卜素的包埋。酪蛋白-葡聚糖接枝共聚物/β-胡萝卜素纳米粒子具有球形外貌,在pH7.0时水合直径约为200nm。该纳米粒子的水溶液具有很高的稳定性,可耐稀释,pH值变化和离子强度变化,可以长时间储存。该纳米粒子也可在干粉状态下保存。包埋在纳米粒子中的β-胡萝卜素可通过胃蛋白酶或胰蛋白酶的水解得到释放。包埋可以提高β-胡萝卜素的抗氧化性,释放后的β-胡萝卜素的反应活性不变。纳米粒子的这些性质使酪蛋白-葡聚糖接枝共聚物可以成为输送低稳定性的疏水性营养物质和药物的载体。第四部分是β-酪蛋白和溶菌酶的自组装行为的研究。我们用两种蛋白,线状的β-酪蛋白和球状的溶菌酶,制备了β-酪蛋白/溶菌酶纳米粒子。当β-酪蛋白和溶菌酶摩尔比为0.4时,两种蛋白在pH3.0-12.0范围内可形成多分散性的β-酪蛋白/溶菌酶静电复合物胶束。在80℃加热处理复合物胶束溶液后,溶菌酶产生凝胶化而β-酪蛋白被陷在凝胶中并阻止了凝胶的宏观聚集,从而形成β-酪蛋白/溶菌酶纳米粒子。该纳米粒子具有球形外貌,它的粒径受到制备条件,诸如pH值以及β-酪蛋白与溶菌酶的摩尔比等的影响。在pH10.0制备的纳米粒子表面有较多的β-酪蛋白分子,而在pH5.0制备的纳米粒子表面有较多的溶菌酶分子。该纳米粒子具有两性,当pH值低于或高于其零电位点时分别带净正电荷或净负电荷。纳米粒子在pH5.0和10.0溶液中可稳定存在,并且疏水性相对较强。纳米粒子表面的净电荷在水溶液中起到稳定纳米粒子的作用。第五部分是β-酪蛋白-葡聚糖接枝共聚物和溶菌酶的自组装行为及其包埋性质的研究。我们通过Maillard反应将葡聚糖接枝到β-酪蛋白上,然后利用β-酪蛋白与溶菌酶的静电相互作用以及溶菌酶的凝胶化性质,制备了表面带有葡聚糖壳层的纳米粒子。纳米粒子的尺寸受到β-酪蛋白-葡聚糖接枝共聚物接枝度的影响,而纳米粒子的组装效率受到β-酪蛋白与溶菌酶摩尔比的影响。纳米粒子具有球形外貌,粒子核的亲水性较强,粒子的水溶液可在生理pH和离子强度条件下长时间稳定。纳米粒子可以包埋阴离子化合物和金颗粒。更多还原

【Abstract】 Micelles composed of amphiphilic copolymers have been explored as carriers for hydrophobic drugs in aqueous solution.Biopolymers are an interesting alternative to synthetic polymers because of their potential loading for both hydrophilic and hydrophobic drugs.In particular,fabricating polymeric carries based on biopolymers via green chemistry process is obviously desirable for the biomedical applications.Caseins are the predominant components in milk and they are a family of phosphorylated proteins.The four casein constituents,αs1-,αs2-,β-andκ-casein, exist in approximate proportions of 3:0.8:3:1 by weight in cow milk and their molecular weights are 19000-25000 Da.All of the four casein molecules in cow milk are amphiphilic proteins and have no defined structure.In food system,casein has many functions,such as emulsification,water binding,fat binding and texturization. These merits endow casein with ideal matrix to fabricate nano-materials for drug delivery.Maillard reaction is a natural,nontoxic reaction which happens during the processing,cooking,and storage of foods.Maillard reaction has been studied extensively which conjugates protein and polysaccharide by linking the reducing end of the polysaccharide to the amines in the protein（terminus and amino groups of lysine）.The products of the Maillard reaction have seldom been used for drug delivery although many applications in other fields have been reported.This thesis focuses on the preparation of the nanoparticles based on casein via green processes and the characterizations as well as the potential applications of the nanoparticles.Two kinds of particles were prepared.One is micelles self-assembled by casein-graft-dextran copolymer,which was prepared through the Maillard reaction. As the core of the micelles is hydrophobic,β-carotene was used as a hydrophobic model compound to study encapsulation and release properties of the micelles.In addition,the Maillard reaction improves the solubility ofβ-casein in acidic solution. The emulsifying activity ofβ-casein-graft-dextran copolymer in acidic solution was studied.Another kind of particles isβ-casein-graft-dextran/lysozyme nanoparticles produced by gelation of lysozyme.The core of the nanoparticles was somewhat hydrophilic.Two kinds of charged derivatives of pyrene and aurichlorohydric acid were used as polar model compounds to study encapsulation properties of the nanoparticles.This thesis contains following five parts: The first part is the study of micellizaion of casein-graft-dextran copolymer.Two natural biomacromolecules,casein and dextran,are used to prepare casein-graft-dextran copolymer through the Amadori rearrangement of the Maillard reaction,a chemical-and solvent-free reaction.The copolymer has a reversible pH-sensitive property:micellization at the pI of casein with a casein core and dextran shell structure and dissociation when pH differs from the pI of casein.The micelles produced at pH 4.6 have a spherical shape and their size is dependent on the grafting degree of the copolymer and the molecular weight of dextran.The micelles are able to encapsulate hydrophobic compounds such as pyrene.The second part is the study of acidic solution properties ofβ-casein-graft-dextran copolymer.β-Casein-graft-dextran copolymer was prepared with the Amadori rearrangement of the Maillard reaction.Dynamic light scattering study shows that at pH range 4.0-5.0 where is close to the isoelectric point ofβ-casein,the copolymers form micelles which are spherical verified by atomic force microscopy imaging.The size and existent time of the micelles depend on the graft degree and the length of dextran side chains of the copolymers.The copolymers with higher hydrophilicity dissolved in molecular state show a better emulsifying ability at pH 4.6 and 8.0.The third part is the study of simultaneous nanoparticle formation and encapsulation driven by hydrophobic interaction of casein-graft-dextran andβ-carotene. Casein-graft-dextran copolymer is soluble in pH 7.0 aqueous solution in molecular state whereasβ-carotene is extremely insoluble.By the method of dialysis or evaporation then dispersing in water,50%ethanol solvent of the copolymer andβ-carotene mixture was changed to 100%aqueous solvent.During this process,the solubility of hydrophobic complex of casein andβ-carotene decreases whereas the solubility of dextran increases gradually,forming the particles with casein andβ-carotene core and dextran shell.The particles have spherical shape and their hydrodynamic diameter is about 200 nm at pH 7.0 solution.The particles can be stored in dried form.The aqueous dispersion of the particles is stable against dilution, pH change,ionic strength change,FeCl₃ oxidation,and long time storage.The encapsulatedβ-carotene can be released by pepsin or trypsin hydrolysis.These characters of the particles provide a possibility for practical applications of the particles to deliver unstable and hydrophobic nutrients and drugs.The fourth part is the study of self-assembly ofβ-casein and lysozyme.Two proteins,linearβ-casein and globular lysozyme,were used to fabricate nanoparticles using a green process.The two proteins formed polydisperse electrostatic complex micelles in the pH range of 3.0-12.0 at the molar ratio ofβ-casein to lysozyme 0.4.β-Casein/lysozyme nanoparticles formed after heating the micelle solution.After a heat treatment at 80℃which is above the denaturation temperature of lysozyme,lysozyme gelated andβ-casein was trapped in the nanoparticles.The nanoparticles have spherical shape and their sizes depend on the pH of the heat treatment,and the molar ratio ofβ-casein to lysozyme.There are moreβ-casein molecules located on the surface for the nanoparticles produced at pH 10.0,whereas more lysozyme on the surface for the nanoparticles produced at pH 5.0.The nanoparticles display amphoteric property:they carry net positive charges and negative charges at pH lower and higher than their zeroζ-potential,respectively.The nanoparticles are stable and relatively hydrophobic at pH around 5 and 10.The net charges on the surface stabilize the nanoparticles in the aqueous solution.The fifth part is the study of the encapsulation properties of nanoparticles selfassembled byβ-casein-graft-dextran copolymer and lysozyme.Dextran was grafted ontoβ-casein through the Maillard reaction and the nanoparticles that have dextran shell were prepared based on the interaction between lysozyme andβ-casein and gelation of lysozyme.The nanoparticle size is influenced by graft degree ofβ-casein-graft-dextran copolymer and the efficiency of the nanoparticle formation is influenced by molar ratio ofβ-casein to lysozyme.The nanoparticles have spherical shape and a somewhat hydrophilic core.The nanoparticle solution is stable against pH change, ionic strength change and long time storage.The nanoparticle can encapsulate anionic compounds and gold particles.更多还原