【作者】 黄丽霞；

【导师】 杨光；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2019， 博士

【摘要】 随着生物医学工程的快速发展,基于天然多糖等生物大分子的功能材料正受到越来越多的关注。壳聚糖是自然界中唯一的天然碱性多糖,具有优异的生物相容性、良好的生物降解性以及多种生物活性,在生物医学领域尤其是组织工程和药物传递系统方面有巨大的应用潜力。本论文围绕壳聚糖微/纳米功能材料的构建及其生物医学应用展开了以下研究工作:(1)多孔壳聚糖微载体的制备及在细胞三维培养中的应用。采用乳化结合热诱导相分离方法(TIPS)制备了多孔壳聚糖微球(CSM)。通过扫描电镜、压汞仪、液体置换等方法对微球的形态和微观结构进行表征,结果显示CSM为直径150～250μm的微球,具有内连通的孔隙结构,孔径为20～50μm,孔隙率高达98%。接触角测试表明CSM具有高亲水性,此外通过原子力显微镜力-位移分析表明CSM具有较高的弹性模量。通过细胞毒性分析和溶血率测试表明CSM具有优异的细胞相容性和血液相容性。肝细胞培养实验表明,CSM不仅能使细胞粘附在微球表面还能使其进入到内部孔隙中生长,细胞保持良好的形态并形成了多方向的细胞连接。通过基因表达、蛋白芯片技术和酶联免疫法进一步分析肝细胞的功能,结果显示肝细胞能在微球上进行正常的合成代谢。以上研究表明,这种壳聚糖多孔微球可作为微载体实现细胞的三维培养,同时维持正常的细胞形态和功能,在细胞治疗、组织工程领域具有潜在的应用价值。(2)智能响应性壳聚糖微载体的制备及在细胞自脱附中的应用。利用β-环糊精(β-CD)与明胶中芳香氨基酸的芳香残基的主客体相互作用,制备了一种基于壳聚糖微球的智能响应性微载体。首先采用乳化交联法制备壳聚糖微球(CSM)并通过化学修饰在微球表面接枝β-CD,然后利用β-CD与明胶之间的主客体作用获得明胶修饰的壳聚糖微球(CSM-g-CD-Gel)。细胞毒性实验表明CSM-g-CD-Gel具有良好的生物相容性。将肝细胞接种于CSM-g-CD-Gel微球上,肝细胞能很好地粘附在微球上生长,培养48 h后,在培养液中加入适量的金刚烷(AD),细胞从微球上自动脱附。这是由于AD与β-CD之间具有更强的亲和力,加入的AD可竞争性地结合β-CD,从而使明胶连同粘附的细胞从微球上脱附。这种响应性微载体避免了传统微载体使用胰酶造成的细胞损伤,该研究建立了一种可大规模培养和获取细胞的新技术。(3)壳聚糖纳米粒子与纳米复合水凝胶的制备及作为药物载体的应用。利用羧甲基-β-环糊精(CMCD)修饰壳聚糖(CS)获得β-CD接枝的壳聚糖(CS-g-CD),并通过三聚磷酸钠(TPP)离子交联组装法制备了粒径约为150 nm的壳聚糖纳米粒子(CS-g-CD NP)。CS-g-CD NP具有良好的p H响应性,在中性条件下保持稳定,而在弱酸性环境中快速解体。利用β-CD与明胶的主客体作用制备了由CS-g-CD NP与明胶组成的超分子纳米复合水凝胶(Gel/CS-g-CD NP)。采用流变仪分析了CS-g-CD NP的含量对Gel/CS-g-CD NP的凝胶动力学性质的影响,结果表明随着CS-g-CD NP含量的增加,Gel/CS-g-CD NP的凝胶温度显著上升,而凝胶化时间迅速缩短,且弹性模量逐渐增加。由于CS-g-CD NP与明胶之间超分子作用的可逆性,Gel/CS-g-CD NP表现出典型的剪切-稀化与自愈合性能。以抗肿瘤药物阿霉素(DOX)为模式药物,制备了载药壳聚糖纳米粒子(DOX-CSCD NP)和载药纳米复合水凝胶(Gel/DOX-CSCD NP)。DOX-CSCD NP和Gel/DOX-CSCD NP均表现出p H响应的药物释放行为,在中性条件下药物释放较慢,而在弱酸性环境中则快速释放药物,且药物释放率增加。体外细胞评价实验表明,载药纳米粒子和水凝胶均具有良好的肿瘤细胞抑制效果,尤其是在弱酸性条件下药物的响应性与持续性释放进一步增强了其抑制效果,有望应用于靶向肿瘤酸性微环境的药物递送系统。综上所述,本论文制备了三种壳聚糖功能材料包括多孔壳聚糖微球、智能响应壳聚糖微球以及壳聚糖纳米复合水凝胶,能作为细胞载体和药物载体进行细胞的三维培养、获取以及药物的靶向可控释放,它们分别在组织工程和肿瘤靶向治疗方面具有潜在的应用价值。更多还原

【Abstract】 With the rapid development of biomedical engineering,functional materials based on biological macromolecules such as natural polysaccharides are receiving more and more attention.Chitosan is the only natural alkaline polysaccharide in nature.It has excellent biocompatibility,good biodegradability,and various biological activities.It has great application potential in biomedical fields,especially tissue engineering and drug delivery systems.This paper focuses on the fabrication of chitosan micro/nano functional materials and their applications in biomedical field.The following aspects of work are included:(1)Preparation of porous chitosan microspheres and their application as microcarriers for 3D cell culture.Porous chitosan microspheres(CSM)were prepared through emulsification coupled with thermally induced phase separation.The morphology and microstructure of the microspheres were characterized by scanning electron microscopy,mercury injection and liquid displacement.The contact angle measurement showed that CSM had high hydrophilicity,and atomic force microscope showed that CSM had high elastic modulus.Cytotoxicity analysis and hemolysis rate test showed that CSM had excellent cytocompatibility and blood compatibility.Hepatocyte culture experiments showed that CSM not only enabled cells to adhere on the surface of microspheres,but also enabled them to enter the internal pores for growth,and the cells maintained good morphology and formed multidirectional cell connections.The function of hepatocytes was further analyzed by gene expression,protein microarray technology and Elisa.The above studies indicated that the as-prepared chitosan porous microspheres could be used as microcarriers to achieve 3D cell culture with normal cellular morphology and functions,which has great application potential in the fields of cell therapy and tissue engineering.(2)Fabrication of smart chitosan microcarriers for responsive cell detachment.Smart and responsive microcarriers based on chitosan microspheres were prepared via the host-guest interaction between β-cyclodextrin(β-CD)and aromatic residues in gelatin.Firstly,chitosan microspheres(CSM)were prepared by emulsion crosslinking approach,the surface of which was then modified with β-CD by chemical grafting.Further the microspheres were modified with gelatin through the host-guest interaction between β-CD and aromatic residues in gelatin.The resultant microspheres were denoted as CSM-g-CD-Gel.Cytotoxicity evaluation showed that CSM-g-CD-Gel had good biocompatibility.High-performance cellular adhesion and growth of hepatocytes were achieved using CSM-g-CD-Gel as microcarriers.More importantly,responsive cell detachment was achieved by adding an appropriate amount of amantadine(AD)in the culture.This is attributed to the higher affinity between AD and β-CD.The competitive binding of AD to β-CD implied gelatin detachment,which led to cell detachment.This trypsin-free cell harvest by the responsive microcarriers could avoid the possible cellular damage and may have bright prospects in cell culture and relevant areas.(3)Preparation of chitosan nanoparticles and their nanocomposite hydrogels with gelatin for p H-responsive drug delivery systems.β-CD-modified chitosan(CS-g-CD)was synthesized by amide reaction between carboxylmethyl-β-cyclodextrin and chitosan.Nanoparticles(CS-g-CD NP)of about 150 nm were then prepared by TPP ionic cross-linking induced assembly.CS-g-CD NP exhibited p H responsiveness,which are stable under neutral conditions,but rapidly disassembled in weakly acidic environment.Supramolecular hydrogels(Gel/ CS-g-CD NP)were prepared by the host-guest interaction between β-CD and aromatic residues in gelatin.The effect of the content of CS-g-CD NP on the hydrogels was analyzed by rheological studies.The results showed that the gelation temperature of hydrogels increased significantly with the increase of the CS-g-CD NP content while the gelation time was shortened rapidly.The elastic modulus of the hydrogels was also increased gradually with the the CS-g-CD NP content.Because of the reversibility of the interaction between CS-g-CD NP and gelatin,Gel/CS-g-CD NP hydrogels exhibited typical shear-thinning and self-healing properties.DOX-loaded CS-g-CD NP(DOX-CSCD NP)and drug-loaded Gel/CS-g-CD NP hydrogels(Gel/ DOX-CSCD NP)were prepared using DOX as the model drug.DOX-CSCD NP and Gel/ DOX-CSCD NP showed p H-responsive drug release behaviors.Drug release was relatively slow under neutral conditions,while it was faster in weakly acidic environment,and the drug release rate increased.In vitro cell evaluation experiments showed that drug-loaded nanoparticles and hydrogels had good inhibition effect against tumor cells,especially under weakly acidic conditions.These p H-responsive drug carriers are expected to be applied in targeting the tumor acidic microenvironment and drug delivery.In summary,this thesis deals with the preparation,characterization and application of three chitosan functional materials including porous chitosan microspheres,smart-responsive chitosan microspheres and chitosan nanocomposite hydrogels,which can be used as cell carriers and drug carriers for three-dimensional cell culture,detachment,and targeted controlled release of drugs.These chitosan-based micro/nano functional materials have potential applications in tissue engineering and tumor-targeted therapy,respectively.更多还原