【作者】 张飞；

【导师】 张洪斌；

【作者基本信息】 上海交通大学 ， 高分子化学与物理， 2014， 博士

【摘要】 糖胺聚糖（又称粘多糖）是动物体内广泛存在的一种多糖，与生物体内许多生理过程有密切联系。对该类多糖的结构、性质、功能化作用研究具有重要的科学意义和应用价值。作为糖胺聚糖家族重要的一员，透明质酸（hyaluronan，hyaluronic acid，HA）具有良好的生物相容性，独特的物化性质以及多样的生理功能。透明质酸以及相关材料已被广泛应用于医药、食品、化妆品等领域。对该多糖基本性质以及基于该多糖的功能材料的构建和应用研究，可以深化对透明质酸本身生理活性和功能化作用的认识，亦能进一步推动相关功能性材料的开发和应用的拓展。透明质酸凝胶化是拓展其作为功能性材料的一个重要的方面。目前对于透明质酸凝胶材料的研究，还主要围绕利用透明质酸本身或者化学改性透明质酸分子上具有的官能团进行化学交联而实现。但是化学交联或衍生化的过程中可能会产生有毒化学物质的残留而使产物具有潜在细胞毒性。这将极大削弱透明质酸天然具有的良好生物相容性。从这一角度，透明质酸物理凝胶的研究就显得尤为重要。此外，将透明质酸应用于纳米材料领域，借助其天然生物大分子特有的一些性质，通过调控、诱导、修饰或者组装制备多样化的纳米结构，并探究所构建功能性纳米材料的应用也是目前备受关注的研究内容。本文基于透明质酸成功制备了一系列（包括物理水凝胶、复合凝胶、复合膜、低维纳米材料和杂化纳米材料等）具有不同结构特性的功能性材料。同时，结合生物活性粘多糖透明质酸和其他组分的各自优势，研究了这些功能新材料在不同领域的潜在应用。主要内容如下：首先采用冷冻解冻法制备了透明质酸物理凝胶，采用流变学、光学显微镜、X射线衍射(XRD)、傅立叶变换红外光谱(FTIR)等一系列表征方法对影响凝胶形成和最终性质的影响因素进行了比较研究，在既有基础上进一步阐明了透明质酸物理凝胶网络分子间作用力模式及其凝胶机理。研究表明，透明质酸冷冻解冻凝胶的形成为典型的物理凝胶化过程。凝胶的形成以及凝胶的性质受诸如凝冷冻解冻过程(冷冻时间，冷冻解冻循环次数等)、透明质酸分子量，以及小分子添加物等诸多因素的影响。长时间的冷冻、多次冷冻解冻循环以及提高透明质酸分子量可以促进凝胶的形成，凝胶的力学性质、产率、热稳定性和抗降解性能均得以提高。对于添加含羧基和羟基的小分子，丁二酸和戊二酸能够参与凝胶网络结构的构筑；而添加草酸、苹果酸、酒石酸以及一些多元醇则会破坏和抑制凝胶网络的形成。低pH值以及低温冷冻条件下所导致的分子链构象变化，以及-COOH参与的有效分子间氢键作用是导致凝胶三维网络结构形成的主要因素。研究还发现，所得酸性和中性凝胶网络结构中分子间氢键作用的形式不同。由酸性凝胶到中性凝胶的中和过程中，由于质子化羧基之间氢键的破坏，使得-COO-和-NHCOCH3参与的氢键在中性凝胶中成为主导的作用力。研究结果也表明所制备的凝胶网络未表现出含有结晶结构的特点，不存在如聚乙烯醇冷冻解冻凝胶中的有序微晶区，而是由多重无规氢键所形成的离散物理交联点将透明质酸分子结合起来而形成的。与透明质溶液相比，所形成的物理交联的透明质酸凝胶具有较好的力学性能、良好的热稳定性和抗酸、抗酶降解性能。采用冷冻解冻方法还制备了一系列透明质酸/聚乙烯醇复合凝胶材料，同时将紫外光照还原法制备的透明酸-纳米银包裹到复合凝胶中得到了纳米复合凝胶。这种纳米复合凝胶是由透明质酸和聚乙烯醇两种聚合物形成的互穿网络结构构成，而纳米银则均匀地分散于基体网络结构中。透明质酸和聚乙烯醇两种组分的配比不同，所得凝胶显示出不同的溶胀特性，且具有一定的pH响应性。采用Kirby-Bauer和LB Broth法对纳米复合凝胶抗菌性研究表明，凝胶对大肠杆菌具有良好的抗菌性。由于透明质酸良好生物相容性、水合性以及对伤口愈合的促进作用，加之纳米银具有的抗菌性，该物理凝胶在临床创伤包缚材料领域有潜在的应用价值。将透明质酸-纳米银（HAS）与血红蛋白（Hb）采用自组装的方法构筑了功能性超薄膜，并对其电化学性质进行了研究。采用透明质酸做为还原剂和稳定，利用紫外光辐射的方法制备了尺度为20～50纳米，表面带负电荷的透明质酸-纳米银颗粒，并通过利用透明质酸纳米银和具有电催化活性的血红蛋白在一定溶液中荷电的不同，采用层层自组装法在玻碳电极表面构筑了透明质酸-纳米银/血红蛋白多层自组装膜([HAS/Hb]n)。对该修饰电极的电化学表征显示：透明质酸-纳米银极大地促进了电极与血红蛋白(Hb)活性中心的电子传递，负载在膜内的Hb在-0.32V有一对对应于亚铁血红素Fe(III)/Fe(II)的准可逆氧化还原峰；负载在自组装膜内的血红蛋白在电极表面具有表面控制的电子传递行为，其非均相电子转移常数为1.0s-1。该自组装膜修饰的电极对氧气和过氧化氢具有较高的电催化活性，由此构筑的传感器对过氧化氢的检测显示出较高的灵敏度和良好的稳定性。合成了带有芘官能团的透明质酸衍生物(芘-透明质酸，Py-HA)，采用包括傅立叶红外光谱、核磁共振谱、紫外-可见光谱、及荧光发射光谱以及表面张力仪等对所合成的两亲性分子及其溶液性质进行了表征。利用这种带有大量π-π共轭结构的衍生物促进诸如石墨、氮化硼以及二硫化钼等片状材料的剥离和稳定，获得了各种形式的碳纳米材料如碳纳米管，石墨烯以及碳纳米洋葱。所制备的表面修饰透明质酸的纳米材料，具有完整的表面结构，在水溶液中显示出良好的稳定性。最后利用两亲性分子芘-透明质酸非共价修饰的氧化还原石墨烯做为多功能性表面平台，采用两种绿色的还原方法(紫外光照和常压下通氢气的方法)构筑了具有不同形貌的不同金属(金，银，钯和铂)的纳米结构。石墨烯表面的芘-透明质酸不仅可以分散和稳定纳米片状碳材料，而且还能够有效地促进金属离子的还原和控制纳米结构的生长。基于不同金属的特性，探究了所制备的杂化纳米材料在传感器方面的应用。金/石墨烯杂化纳米材料修饰的玻碳电极对过氧化氢具有较高的电化学催化活性，以此构筑的传感器具有较宽的线性范围、较低的检测限以及较快的检测响应。钯/石墨烯杂化纳米材料在不同体积分数的氢气存在下显示出不同导电性，以此构筑了氢气传感器。所构建的传感器由于杂化纳米材料所具有的不同结构而具有不同强度的响应性。更多还原

【Abstract】 Mucopolysaccharide or Glycosaminoglycan is widely distributed inanimals’body and presents multiple physiological functionalities. Hyaluronan (HA) isone of the most representative mucopolysaccharides and supposed to be the onlypolysaccharide that exists in almost all animal species, from bacteria to human beings.The biological functions of HA include maintenance of the elastoviscosity of liquidconnective tissues such as joint synovial and eye vitreous fluid, control of tissuehydration and water transport, supramolecular assembly of proteoglycans in theextracellular matrix, and numerous receptor-me-diated roles in cell detachment,mitosis, migration, tumor development and metastasis, and inflammation. Moreover,as one natural biopolymer, it is inherently biocompatible, biodegradable, bioactive,non-immunogenic and nonthrombogenic. For these merits of HA, this bioactivepolysaccharide has been an attractive building block for the fabrication of functionalmaterials. Nowadays, HA and the materials based on it are widely used in the fields ofpharmaceutical, food and cosmetics, tissue engineering and nanomaterials, etc.Among the stuies of HA hydrogels most work focused on the chemicallycrosslinked hyalruoan or HA derivatives. In the process of synthesizing HA hydrogelsby chemical reaction, the use of crosslinking agent or organic solvent, and theexistence of reacting by-products in final hydrogels are inevitable. These will impairthe biological compatibility in both short and long-term applications of HA, especiallyin the biomedical aspects. Therefore, the study on the physically crosslinked HAhydrogels will be very important and promising. Moreover, HA can be used asbioactive component for construction of nanomaterials, which has gained researchers’enormous concern and interest. The main work in this dissertation thus revolves around the construction of hyluroan-based functional materials with various nano-andmicro-structures and different potential applications. HA in these processes acts asimportant building block for creating materials and presents multiple functionalitieswith regulating the nanostructure and promoting the biocompatibility etc. The maincontents and conclusions are summarized as follows:Firstly, physically crosslinked hydrogels from HA were prepared by freeze-thawtechnique. The effects of processing steps (freezing time and number of freeze-thawcycles), molecular weight of HA, and some small molecular additives such asdicarboxylic acids and polyols as probes on the formation of the HA cryotropichydrogels were investigated by dynamic rheometry, optical microscopy, X-raydiffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and gelpermeation chromatography (GPC). The experimental results showed that the longperiod of the freezing time and repeated freeze-thaw cycles benefited the alignment ofthe polymer chains in the unfrozen polymer phase and thereby promoted theformation of intermolecular aggregations and densely fibrillar network structures. HAwith a higher molecular weight was favorable to the formation of HA cryogel with astronger mechanical performance. The influences of various small molecular additiveson the gelation of HA had relevance to the intermolecular associations in the gelnetwork. Experimental findings showed that both succinic acid and glutaric acid werecapable of participating in the formation of HA cryogel whereas the addition of oxalicacid, malic acid and tartaric acid as well as some polyols (glycol, butanediol andglycerol) inhabited the gelation of HA. Protonation of the polyanion of HA at properlow pH served as a prerequisite of the association of multiple interchain hydrogenbonding among groups of-COOH and-NHCOCH3and the formation of networkafterwards. HA cryogels showed fibrillar network in which HA chains were associatedinto bundles. It was reavled that the contribution of the crystallization of HA was notobvious and the gel formation was essentially related to the HA conformation changesand the molecular aggregations driven by the hydrogen bonds among–COOH and-NHCOCH3groups. While in HA acidified cryogels hydrogen bonding between-COOH played dominating roles the in stabilizing the network, hydrogen bonding involving–COO-and NHCOCH3became the main driving force stabilizing thenetwork in neutral cryogels. All the experimental results in the present stronglysuggested that in either acidified or neutral cryogels hydroxyl groups of HA made nonoticeable contributions to such hydrogen bonding in the network. In comparison withnative HA in aqueous solution at the same concentration, the neutral HA cryogelsobtained by in situ neutralizing the acidified HA cryogel, showed higherviscoelasticity, better resistances to the acidic decomposing and enzymaticdegradation.Secondly, novel nanocomposite hydrogels composed of HA, poly(vinyl alcohol)(PVA), and silver nanoparticles were prepared by several cycles of freezing andthawing. The nanocomposite was then characterised using FTIR, differential scanningcalorimetry (DSC), XRD, and scanning electron microscopy (SEM). The complexhydrogels consisted of semi-interpenetrating network structures, with PVAmicrocrystallines as junction zones. By increasing the HA content, the crystallinityand melting temperature of the complex hydrogels decreased, whereas the glasstransition temperatures of these materials increased because of the steric hindrance ofHA and the occurrence of intermolecular interactions through hydrogen bondingbetween HA and PVA in the complex hydrogels. Swelling studies showed that thoseof the complex hydrogels can be significantly improved in comparison with theswelling properties of the cryogels from PVA alone and presented a pH-sensitivemanner. In addition, silver nanoparticles were synthesised through UV-initiatedphotoreduction with HA functioning as a reducing agent and stabiliser. The silvernanoparticles were then incorporated into the HA/PVA complex hydrogel matrix. Thesize and morphology of the as-prepared Ag nanoparticles were investigated throughUltraviolet-visible light spectroscopy, transmission electron microscopy, XRD, andthermogravimetric analysis (TGA). The experimental results indicated that silvernanoparticles20nm to50nm in size were uniformly dispersed in the hydrogel matrix.The antibacterial effects of the HA/PVA/Ag nanocomposite hydrogel againstEscherichia coli were evaluated by Kirby-Bauer and LB Broth method. The resultsshowed that this nanocomposite hydrogel possessed high antibacterial property and has a potential application as a wound dressing material.In the third section, HA-silver nanoparticles (HSNPs) were prepared byUV-initiated photoreduction and protein hemoglobin (Hb) was then alternatelyassembled with the prepared negatively charged HSNPs into layer-by-layer (LBL)films on solid surface. The electrochemical behavior and electrocatalytic activitiestoward oxygen and hydrogen peroxide of the resulting films were studied. It wasfound that the HSNPs greatly enhanced the electron transfer reactivity of Hb as abridge between protein and electrode and that the assembled films showed a pair ofnearly reversible redox peaks with a formal potential of0.32V (vs. Ag/AgCl) for theheme Fe(III)/Fe(II) redox couple. The immobilized Hb in the films maintained itsbiological activity, showing a surface controlled process with a heterogeneouselectron transfer rate constant (ks) of1.0s-1and displaying the same features of aperoxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide. Thiswork provides a novel model to fabricate LBL films with protein, polysaccharide andnanoparticles and may establish a foundation for fabricating new type of biosensorsbased on the direct electron transfer of redox proteins immobilized in nanocompositemultilayer films with underlying electrodes.Fourthly, pyrene-conjugated HA (Py HA) was synthesized and characterized byATR-FTIR, NMR, UV-vis, flurorecent spectroscopy and surface tension. Py HAfacilitated the exfoliation of low-dimensional nanomaterials including graphene,hexagonal boron nitride (h-BN), molybdenum disulfide (MoS2), carbon nanotubes(CNTs) and carbon nano-onions (CNOs) in water (and PBS solutions), with theassistance of sonication. The materials were stabilised by the Py HA as uniformlydispersed suspensions in water, with excellent crystallinity. Moreover, thefunctionalized hybrid nanomaterials had biocompatibility factored in, and offeredscope for applications in biomedical fields, and beyond.And finally, we have developed a versatile platform involving reduced grapheneoxide and amphiphilic Py HA for fabricating composite nanomaterials involvingnoble metals (Au, Ag, Pd and Pt) using facile and green methods, with UV irradiationor introduction of hydrogen gas as the benign reducing process/agent. Py HA not only acted as a stabilizing agent for RGO, but also facilitated and controlled thedecoration of the metal on the Py-HA-RGO substrate. The hybrid nanocomposites canserve as sensing material for different analytes, depending on the specific propertiesof metals. The Au-graphene hybrid material was employed as the electrochemicalenhanced material for H2O2sensing, with wide linear ranges and low detection limits,and presented different sensing behaviours depending on the different nanostructures.The Pd-graphene hybrid material gave a change in conductivity in the presence ofhydrogen gas when assembled in an interdigitated electrode, which was the basis of ahydrogen gas sensing.更多还原