Hydrogel Preparation, Chitosan Modification and Application

水凝胶用作创口敷料时有很多的优点。壳聚糖是天然的阳离子多糖,已经在很多领域得到了应用。我们制备了几种具有抑菌能力的水凝胶敷料，同时利用可生物降解聚酯和多聚肽对壳聚糖进行了修饰，得到了一些新的结果。 本论文的工作主要包括以下两部分： 一.含有抗生素或纳米银粒子的水凝胶的制备和表征。 1．载药聚乙烯醇/聚乙烯基吡咯烷酮/壳聚糖（PVA-PVP-Chitosan）复合水凝胶的制备和抑菌性能评价。采用循环冷冻－熔融与辐射交联相结合的方法制备出了PVA-PVP-chitosan复合水凝胶，测定了凝胶的溶胀率、凝胶含量、力学强度和结晶性能，并考察了所用壳聚糖分子量，冷冻－熔融循环处理次数和辐射剂量对凝胶性能的影响。然后通过溶胀法将抗生素（环丙沙星）和壳寡糖载入水凝胶中，考察了它们的体外释放行为。同时利用琼脂糖平板法检验了载药凝胶的抑菌能力，结果表明它们可以有效抑制革兰氏阳性菌和革兰氏阴性菌的繁殖和生长。 2．含纳米银-PVA-PVP水凝胶的制备和抑菌性能评价。通过循环冷冻－熔融法制备了含银的PVA-PVP复合水凝胶。以含银的PVA-PVP复合膜为模型，利用DSC、红外光谱、紫外－可见光谱和X-射线衍射考察了掺入的纳米银与PVA-PVP网络的相互作用。利用扫描电镜观察了PVA-PVP凝胶的网络结构和银纳米粒子在网络中的分布。利用琼脂糖平板法和LB肉汤法对含银PVA-PVP凝胶的抑菌性能进行了评价，结果证明这种水凝胶可以有效抑制大肠杆菌和金黄色葡萄球菌的生长和繁殖。 二. 壳聚糖的改性和应用。 1．发展了一种制备聚ε－己内酯（PCL）接枝壳聚糖共聚物的新方法，并且由合成的接枝共聚物在水中自组装得到纳米粒子。利用DLS、AFM和SEM对纳米粒子进行了表征，结果显示制备的纳米粒子为圆球形或椭圆形。利用茚三酮显色法准确测定了活性胺基的含量，发现纳米粒子表面活性胺基的数量可以通过改变PCL接枝度进行调节。 2．由6-O-三苯甲基壳聚糖引发赖氨酸NCA的开环聚合得到了聚赖氨酸（PLL）接枝壳聚糖阳离子共聚物。利用红外光谱、核磁共振和GPC对接枝共聚物的分子结构和分子量进行了表征。通过动态光散射测量发现接枝共聚物可以和小牛胸腺DNA形成粒径在100~340 nm之间的复合物。凝胶阻滞电泳证明接枝共聚物对质粒DNA的结合和保护能力要优于壳聚糖。细胞转染实验证明接枝共聚物对293T和Hela细胞的转染效率要远远高于壳聚糖原料和PLL。同时，接枝共聚物的转染效率对PLL的聚合度有明显的依赖性，增加PLL聚合度有助于提高接枝共聚物的转染效率。这种新型的阳离子接枝共聚物有望用作高效基因载体。Hydrogels have many advantages when used as wound dressing. Chitosan is a naturally occurring cationic polysaccharide, which has been utilized in many fields. We have prepared several kinds of hydrogels with antibacterial ability, and modified chitosan with biodegradable polyester and polypeptides. We obtained some new results in following aspects: The first part was concentrated on preparing antibiotic or silver nanoparticles medicated hydrogels. Poly(vinyl alcohol)/poly(N-vinyl pyrrolidone)/chitosan hydrogels were prepared by repeated freeze-thaw treatments and subsequent γ-ray irradiation and then medicated with ciprofloxacin lactate and chitosan oligomer. The gel content, swelling ratio, tensile strength and crystallinity of the hydrogels were determined. The effects of chitosan molecular weight, freeze-thaw treatment procedure and radiation dosage on the hydrogel properties were examined. The ciprofloxacin lactate and chitosan oligomer were soaked into the hydrogel. Their in vitro release from the hydrogel was examined. The hydrogels were proved to be of excellent antibacterial ability. Silver nanoparticles filled PVA-PVP hydrogels were prepared by freezing and thawing method. The influence of silver nanoparticles on the properties of PVA-PVP matrix was investigated through DSC, FT-IR, UV-Vis spectroscopy and X-ray diffraction, using silver particles filled PVA-PVP films as models. The morphology of freeze-dried PVA-PVP hydrogel matrix and dispersion of silver nanoparticles in the matrix were examined by SEM. The antibacterial effects of silver filled PVA-PVP hydrogels against Escherichia coli and Staphylococcus aureus were examined by film attachment method and LB broth method also, it is proved that the silver filled hydrogels had an excellent antibacterial performance. The second part was concentrated on chitosan modification and application. A novel synthetic approach to biodegradable amphiphilic cationic copolymers based on PCL and chitosan was presented, and the prepared copolymers were used to prepare nanoparticles successfully. The nanoparticles made from the graft copolymers were investigated by DLS, AFM and SEM measurements. It was found that the copolymers could form spherical or elliptic nanoparticles in water. The amount of available primary amines on the surface of the prepared nanoparticles was evaluated by ninhydrin assay, and it can be controlled by the grafting degree of PCL. Poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring opening polymerization (ROP) of ε-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the cationic copolymers and calf thymus DNA was measured by DLS. It was found to be in the range of 120~340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection efficiency of the copolymers in 293T and Hela cells was evaluated. The results showed that the gene transfection ability of the copolymer was better than that of the starting material chitosan, and it was dependent on the degree of polymerization of PLL. The PLL-g-Chi copolymers could be used as effective gene delivery vectors