天然大分子透明质酸(hyaluronic acid，hyaluronan，HA)是由D-葡萄糖醛酸和N-乙酰基葡萄糖胺二糖重复单元组成的线性聚阴离子多糖。HA被认为是唯一几乎存在于从细菌到人类所有动物体之中的粘多糖。人体细胞外基质以及细胞表面均含有一定量的HA。由于HA具有生物相容性、生物可降解性、非免疫性、靶向性和高持水性及粘弹性等特点，很多基于HA的生物材料得以被开发并在生物医药领域被广泛应用，如作为人工关节液、术后防粘连材料、主动靶向递送成像和抗癌剂的有效载体材料以及聚电解质组装膜等。本论文基于HA的固有特性以及化学修饰的易操作性，构建和开发了一系列基于HA的生物材料，研究了它们在骨关节炎治疗、术后防黏连、抗癌药物机铱配合物和多柔比星的靶向释放，以及酶固定法合成结构脂质等生物医药领域中的诸多应用。以下为具体研究内容和研究结果。 1)基于点击化学，设计并制备了一类用于骨关节炎治疗的长效粘弹性补充剂。 HA是正常膝关节中滑液的主要成分，并为滑液提供粘弹性，但在罹患骨关节炎的关节中，滑液因HA的降解，失去了其独特的粘弹性和润滑性。在骨关节炎患者膝关节内外源性注射HA，目前被认为是恢复滑液粘弹性和润滑能力的最重要的有效方法。然而，HA的体内快速酶降解特性极大影响了疗效。为克服HA酶降解快的问题，本文先采用乙烯基砜改性HA，再将其与二硫醇封端的聚乙二醇交联得到HA-VS／SH-2-PEG微凝胶，然后将微凝胶混合添加至HA溶液中，制备了一系列具有可调节粘弹性和可控降解速率的HA可注射流动水凝胶。该粘弹性补充剂不仅表现出高抗酶降解性能力，还具有高粘弹性和良好的润滑效果以及高效的载药能力。动物实验显示，所制备的粘弹性补充剂具有良好的生物相容性，而且用粘弹性补充剂治疗的膝关节具有抑制体内骨关节炎进展的能力。肉眼和组织学观察表明，用粘弹性补充剂治疗兔骨关节炎的宏观评估分值小于1.85。该研究结果为采用可注射流动水凝胶治疗骨关节炎提供了有效策略，也为临床应用提供了技术支持。 2)基于冷冻解冻法，不采用任何化学交联剂，制备了用于防术后黏连的物理水凝胶。腹部和盆腔手术后腹膜粘连很常见，会导致各种严重的并发症。尽管已有许多药物和屏障装置被开发以达到最小化或防止术后粘连目的，但临床功效并不十分令人满意。本工作中，开发了一种可生物降解和生物相容的HA冷冻解冻物理凝胶，并研究了该HA水凝胶在预防术后腹膜粘连方面的功效。体外细胞试验表明， HA冷冻解冻凝胶具有优异的生物相容性。此外，采用侧壁缺损-盲肠磨损的大鼠模型评估了该凝胶的抗粘附功效。研究表明，该凝胶能有效预防术后腹膜粘连，且无毒副作用。 3)制备和构建了高水溶性、pH和还原反应药物双重响应性的铱类抗癌药物靶向递送系统。含有π-键合的联苯基四甲基环戊二烯基(Cp〓)、CAN-螯合的苯基吡啶(phpy)和吡啶(py)配体的有机铱配合物(Ir(IlI))[(η⁵-Cp〓)Ir(phpy)(py)] PF₆具有对癌细胞更有效的抗肿瘤活性。然而，不良的位点特异性递送、低溶解度和低生物利用度是化疗抗癌药物的常见不足。为克服这些不足，通过制备含有二硫键的HA-胱胺-芘基(HA-ss-Py)和HA-吡啶(HA-Py)两亲性HA胶束(粒径为250～260 nm)，将Ir(III)药物加载于到这两种两亲性胶束中并形成纳米颗粒(载药量为33.5～36.4％)，成功制备了用于负载Ir(III)药物的、具有CD44靶向性、 DH和还原反应药物双重响应性的递送系统。研究表明，HA-ss-Py形成的胶束在生理条件下足够稳定，但胶束在还原环境(20mM谷胱甘肽(GSH))中又可快速解离(释放率达到95％)。A549癌细胞可有效地内吞该胶束粒子。A549细胞的体外细胞毒性测定证明，负载Ir(III)的HA基纳米颗粒具有比游离Ir(III)抗癌药物更高的细胞毒性，其中负载Ir(III)的HA-ss-Py纳米颗粒具有更强的肿瘤抑制作用(肿瘤抑制率达到92％)。相应的生物分布实验也表明，其在肿瘤细胞中有更多累积，更有潜力用作靶向癌症治疗Ir(III)抗癌药物载体。 4)制备并研究了基于空心介孔二氧化硅纳米粒子(HMSNs)，含有二硫键，硼酯键和HA的新型药物释放系统(HMSNs-NH₂-CBA-DTPA-HA)。该新型药物释放系统，是通过采用二硫键和硼酯键作为还原和pH双重响应型断裂键，以及HA作为“gatekeeper”的构建策略，将抗癌药物多柔比星(DOX)递送至靶向肿瘤细胞并迅速释放(载药率12.5％，释放率达到80.7％)。体外药物释放实验及生物学评价实验显示，负载DOX的HMSNs-NH₂-CBA-DTPA-HA纳米颗粒具有pH和氧化还原双响应性药物释放性能和肿瘤靶向性，并能高效诱导肿瘤细胞凋亡。 5)制备了透明质酸(HA)和壳聚糖(CHI)修饰的磁性微球(Fe₃O₄@SiO₂@{CHI／HA}₃，630 nm)，并将其用于脂肪酶的固定和功能性结构脂质的高效合成。分别采用水热反应和溶胶-凝胶法，首先合成了Fe₃O₄磁性微球和Fe₃O₄@SiO₂核／壳微球，再采用LbL技术将多糖大分子修饰于微球表面，最后通过利用1-乙基-3-(3-(二甲基氨基)-丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)偶联法，将脂肪酶固定到磁性载体的表面上。固定化率达48.6 ㎎／g。通过多种分析表征手段(FT-IR、TEM、SEM-EDS、元素分析、VSM、TGA、以及XRD)，详细研究了载体和固定化脂肪酶的形态、核壳结构和磁性能。研究表明，所构建的磁性微球Fe₃O₄@SiO₂@{CHI／HA}₃具有优异的脂肪酶固定性能，可用于高效合成具有重要营养价值的结构脂质1,3-二油酰-2-棕榈酰甘油(OPO)。磁性固定化脂肪酶表现出良好的热稳定性(热处理48小时后， Fe₃O₄@SiO₂@{CHI／HA}₃@lipase仍能保留其初始活性的53.9％)、长期储存稳定性(储存30天后，Fe₃O₄@SiO₂@{CHI／HA}₃@lipase仍然保留了其初始活性的78％)和可重复利用性(可重复使用9次)，是一类可用于结构脂质高效合成的绿色生物催化剂。 关键词：透明质酸，骨关节炎，术后防粘连，药物释放，酶固定

 Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of D-glucuronic acid and N-acetyl-Dglucosamine. It is evolutionarily conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Recently, a variety of biological functions of HA have been explored and a number of customized applications have been investigated taking advantages of the interaction between HA and biological tissues. As a naturally-occurring polysaccharide, HA has been used for joint lubrication and prevention of peritoneal adhesion in its intact form due to the excellent biocompatibility, viscoelasticity, biodegradability, and hygroscopic properties. HA can be easily functionalized via the chemical modification of its carboxyl and hydroxyl groups. In addition, the interaction of HA with the CD44 receptor, whose expression is elevated on the surface of many types of tumor cells, makes this polymer a promising candidate for intracellular delivery of imaging and anticancer agents exploiting a receptor-mediated active targeting strategy. In recent years, HA has become key candidate for the preparation of HA-based PEMs using the LbL assembly technique mostly due to its tunability through assembly conditions, hydrophilicity, biocompatibility, bioactivity, and biodegradability. Thus, taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, we have developed various HA-based biomaterials with promising and broad potential in biomedical field. HA is a major component of synovial fluid in normal knee joints and provides viscoelastic properties to synovial fluid. In osteoarthritic joints, synovial fluid loses its unique viscoelastic and lubrication properties. Intra--articular injection of HA in osteoarthritic knee joints is believed to restore viscoelasticity and lubrication abilities to synovial fluid. However, the main drawback of HA is its rapid degradation in synovial fluid. In order to overcome the problem of rapid clearance of HA, an HA-based hydrogel (vinyl sulfone-modified HA crosslinked by dithiol-terminated poly(ethylene glycol), HA-VS/SH-2-PEG) was synthesized in this study. Injectable fluid hydrogels (viscosupplements) possessing tunable viscoelasticity and controllable degradation rates were then prepared by adding HA-VS/SH-2-PEG microgels to HA solutions. The resultant viscosupplements exhibited enhanced viscoelastic properties, lubrication effects, efficient triamcinolone acetonide payload capacity, and high resistance to enzymatic degradation (45% of initial storage modulus) along with good biocompatibility and the capability to inhibit the progression of osteoarthritis in vivo. The average scores for macroscopic assessment of rabbits treated with viscosupplements (HV-1, HV2, HV-3) were 1.85,1.75, and 1.75, respectively. Overall, the results provided support for the use of injectable fluid hydrogels as a strategy for the treatment of osteoarthritis and potential for the clinical translation. Peritoneal adhesion is very common after abdominal and pelvic surgery, which leads to a variety of severe complications. Although numerous pharmacological treatments and barrier-based devices have been investigated to minimize or prevent postoperative adhesion, the clinical efficacy is not very encouraging. In this work, a biodegradable and biocompatible HA cryogel was developed and the efficacy of HA cryogel in preventing postoperative peritoneal adhesion was investigated. In vitro cell tests showed that HA cryogel was non-toxic. Moreover, the rat model of sidewall defect-cecum abrasion was employed to evaluate the anti-adhesion efficacy of the HA cryogel. The results demonstrated that HA cryogel could effectively prevent postoperative peritoneal adhesion without side effects. The combination of biocompatibility, appropriate biodegradation period, and excellent postoperative anti-adhesion efficacy make HA cryogel a promising candidate for the prevention of postsurgical peritoneal adhesion. The organoiridium complex (Ir(III)) [(η⁵-Cpxbiph)Ir(phpy) (py)]PF₆ containing π-bonded biphenyltetramethylcyclopentadienyl(Cp〓), C〓N-chelated phenylpyridine (phpy), and pyridine (py) ligands has more potent antitumor activity as a new generation of drug than cisplatin toward various cancer cells. However, poor site-specific delivery, low solubility, and poor tumor penetration are common limitations of chemotherapy drugs. To develop CD44-targetable, pH-, and reduction-responsive drug delivery systems for Ir(III) drugs, the amphiphilic hyaluronan (HA)-based conjugates of HA-cystamin-pyrenyl (HA-ss-Py) containing disulfide bonds and HA-pyrenyl (HA-Py) were designed (mean diameter 250-260 nm). The Ir(III) drug was readily loaded into these two amphiphilic conjugates and nanoparticles were formed with 33.5～36.4% drug loading content. Dynamic light scattering (DLS) studies showed that the micelles formed from HA-ss-Py were sufficiently stable under physiological conditions, but were prone to rapid dissociation in reducing environments (20 mM glutathione (GSH)). In subsequent confocal microscopy analyses, A549 cancer cells efficiently internalized HA-based micelles. Moreover, in vitro cytotoxicity assays in A549 cells demonstrat that Ir-loaded HA-based nanoparticles have higher cytotoxicity (tumor inhibition rate of 92%) than the free Ir(III) anticancer drug. Finally, systemic administration of Ir(III)-loaded HA-ss-Py nanoparticles enhanced tumor inhibition in vivo, and the corresponding biodistribution experiments showed that HA-ss-Py micelles accumulate in tumors. Overall, our results suggest that HA-ss-Py micelles have a great potential to be used as an effective Ir(III) drug carrier for targeted cancer therapy. In this study, a novel drug delivery system (HMSNS-NH₂-CBA-DTPA-HA) based on hollow mesoporous silica nanoparticles (HMSNs) was developed for delivering anticancer drugs (e.g., doxorubicin (DOX), drug loading content of 12.5%) to targeted tumour cells by using disulfide bonds and boronic acid-catechol ester bonds as redox and pH-sensitive linkers and hyaluronic acid (HA) molecules as both capping and targeting agents. Detailed physiochemical characterization further demonstrated that HMSNS-NH₂-CBA-DTPA-HA has been successfully constructed. The in vitro drug release experiments displayed the pH and redox dual-responsive and sustained drug release properties of DOX loaded HMSNs-NH₂-CBA-DTPA-HA (drug release of 80.7%). Additionally, a series of biological evaluations indicated that these DOX loaded HMSNS-NH₂-CBA-DTPA-HA could accurately target A549 cells to induce cell apoptosis in vitro. These results demonstrated that DOX loaded HMSNS-NH₂-CBA-DTPA-HA was suitable as a potential and efficient drug delivery nanosystem for cancer therapy. A promising strategy for lipase immobilization based on the natural polymers of polysaccharides (hyaluronan (HA) and chitosan (CHI)) functionalized magnetic microspheres (Fe₃O₄@SiO₂@{CHI/HA}₃, 630 nm) was developed. First, Fe₃O₄ magnetic microspheres and Fe₃O₄@SiO₂ core/shell microspheres were synthesized by hydrothermal reaction and sol-gel method, respectively. Owing to the abundant carboxyl groups in HA chains, the lipase was covalently bonded on the surface of the polysaccharide functionalized magnetic supports by ultilizing l-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry to produce robust biocatalysts of Fe₃O₄@SiO₂@{ CHI/HA }₃@lipase. The morphology, core-shell structure, and magnetic property of the supports and immobilized lipase were investigated through various analytical techniques, including FT-IR analysis, TEM, SEM equipped with energy dispersive spectrometer (SEM-EDS), elemental analysis, vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). Consequently, the magnetic Fe₃O₄@SiO₂@{CHI/HA}₃ microspheres exhibited a superior performance in terms of immobilizing lipase (48.6 ㎎/g). The magnetic immobilized lipase showed good thermal (retained 53.9% of its initial activity at 60℃ after 48 h) and long-term stability (retained 78% of its initial activity after 30 days at 4℃), and reusability (nine consecutive cycles) for the synthesis of the structured lipid of l,3-dioleoyl-2-palmitoylglycerol (OPO), which has recently received much interest as a healthy component of food, oil, and pharmaceutical intermediates. The magnetic immobilized lipase could be considered a green and sustainable biocatalyst for the highly efficient synthesis of OPO. Key words: Hyaluronan, Osteoarthritis, postoperative adhesion prevention, drug delivery, enzyme immobilization.