壳聚糖/端羧基呫吨酮自组装复合纳米粒子及其协效抗菌机制

As the natural antibacterial material, the application of chitosan was limited by the low antibacterial effect, the poor solubility and the weak dispersion, so it is important to search for the new kind of simple, convenient and effective way. According to composite compensation effect, the antibacterial composite nanoparticles will be fabricated by self-assembly of chitosan and carboxyl terminated xanthones through electrostatic interactions and hydrophobic interactions. The nanocomposite could exert influence on the inner and the outer space of the bacteria, respectively. And the dispersion and the antibacterial property in the wide range of pH should be obviously improved. In this project, the influence of the substituents position on the polyhydroxy xanthones will be investigated; the effects of molecular weight, deacetylation degree on the interfacial interactions between chitosan and xanthones, the physicochemical properties and the morphology of the nanoparticles will be characterized by SEM, TEM, DLS, FTIR, DSC and XPS. Moreover, the antibacterial performance of the nanoparticles against gram positive bacteria and gram negative bacteria will be studied by the haloes methods and oscillation flask method, while the relationship between the structure and the antibacterial property will be investigated. In addition, the synergistic antibacterial mechanisms will be revealed from the micro-structure analysis by SEM, TEM, fluorescence microscope and spectroscopy. The achievement of the project may not only offer important fundamental data, theoretical proof for new natural chitosan antibacterial materials, but also afford new ideas for the similar antibacterial material.

壳聚糖作为天然抗菌材料，存在抗菌活性低、水溶性差、分散性差等缺点，限制了其进一步应用。本项目拟利用复合相补效应，将壳聚糖和端羧基呫吨酮通过静电和疏水/亲水相互作用自组装成复合纳米粒子，分别从细菌内部和外部抗菌，以提高其分散性和较宽pH范围内的抗菌性，从而拓宽其应用。本项目主要研究内容是：研究取代基位置和官能团对端羧基呫吨酮抗菌性能的影响；通过扫描电镜、红外光谱、粒度仪及X射线光电子能谱等研究壳聚糖分子量、脱乙酰度等对壳聚糖/呫吨酮复合纳米材料的界面结合作用，及该作用对粒径、形貌、水溶性和稳定性等物理化学性质的影响；采用晕圈试验法、振荡瓶法研究材料对革兰氏阴性菌和革兰氏阳性菌的抗菌性能，探索材料结构和抗菌性能的关系；采用电镜、荧光显微镜并结合光谱法和探针试剂，从超微观结构角度揭示材料对细菌内外的双重抗菌机制。本项目的顺利实施，可为新型壳聚糖天然抗菌材料的研究提供重要的基础数据和理论依据。

壳聚糖由于来源广、生物相容性好以及抑菌杀菌性能，是理想的天然高分子抗菌材料；但它抗菌活性低，水溶性差，限制了其进一步应用。为了解决上述问题，本项目主要研究了壳聚糖和呫吨酮复合材料的制备方法和抗菌性能，并通过各类仪器和方法对相关合成材料的化学结构、形貌、粒径和晶型等进行表征，研究了抗菌机理。具体研究内容和成果如下：（1）合成了几种多羟基端羧基呫吨酮，研究了取代基对呫吨酮抗菌性能的影响，抗菌强弱顺序为1-羟基-3-氧乙酸基吨酮>1, 3-二羟基吨酮 >1, 3, 6-三羟基吨酮，其中，1-羟基-3-氧乙酸基吨酮对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度（MIC）分别为250和500 µg·mL-1。（2）通过酯化反应将不溶的1, 3-二羟基吨酮接枝到羧甲基季铵化壳聚糖上得到了一种水溶性的壳聚糖聚合物，它对大肠杆菌和金黄葡萄球菌的MIC均为125 µg·mL-1。（3）通过静电自组装制备了分散性、稳定性良好的端羧基呫吨酮/季铵化壳聚糖（CXan/TMC）静电自组装复合材料，筛选出最佳制备摩尔比为1:4，平均粒径为174 nm，季铵化壳聚糖（TMC）和呫吨酮(CXan)之间通过静电作用和氢键作用结合；材料对大肠杆菌和金黄葡萄球菌的MIC为100和25 µg·mL-1，比TMC和CXan的MIC减少了75%。（4）利用精氨酸对壳聚糖进行酰胺化改性，制备出可溶于水的精氨酸化壳聚糖（ACS），将其与1-羟基-3-氧乙酸基呫吨酮（HOAX）静电自组装，获得了抗菌性和分散性良好的复合抗菌材料(HOAX/ACS)，其最佳制备摩尔比为1:4，对大肠杆菌和金黄葡萄球菌的MIC分别为62.5、125 µg·mL-1，比HOAX和ACS都有显著提高。本项目对开发壳聚糖基天然抗菌材料和植物中提取的天然生物活性小分子在抗菌领域的应用具有指导意义。