摘要
由于多种细菌性疾病的出现,研究者们对高效广谱抗菌材料的研发产生了浓厚的兴趣。目前,已发现的具有抗菌活性的物质有次氯酸钠、臭氧、二氧化氯、金属离子、季铵盐、季磷盐、多肽、胍盐、N-卤胺等。其中,N-卤胺抗菌材料由于其独特的性质已受到学术界的高度重视。N-卤胺抗菌材料因具有抗菌性强、稳定性高、易保存、可再生、无腐蚀、无毒、廉价等优点,广泛应用于医疗器械、医院、水净化系统、食品包装、食物保鲜、卫生设施等多个领域。N-卤胺抗菌材料的抗菌机理为通过释放氯离子,在细菌细胞表面发生离子交换反应,从而破坏或阻止细胞正常的新陈代谢作用,促使细菌死亡。对于N-卤胺而言,接触面积对其抗菌性能至关重要。缩小N-卤胺抗菌材料的尺寸能增大其比表面积,能为材料提供更多的抗菌性官能团,有效官能团的增加可以提高材料的抗菌效率。
纳米材料在尺寸上从几个纳米到百余纳米,能呈现出比传统材料更加优越的物理、化学、力学等性能。由于其小尺寸效应和表面界面效应,纳米材料具备巨大的应用潜力和良好的发展前景。因此,通过制备纳米尺寸的N-卤胺抗菌材料来增大其比表面积是提高抗菌效率的有效方法。但是,纳米尺寸的N-卤胺抗菌材料也存在不足之处,即回收过程过于繁琐。抗菌操作通常在水溶液中进行,且操作结束后须通过分离程序来处理残留的抗菌材料,而从大量的水溶液中分离出具有纳米尺寸的N-卤胺抗菌材料需要较高的成本。
磁性纳米粒子由于其独特的性质已广泛应用于材料的磁分离技术中。磁分离技术是利用外加磁场对磁性或者易受磁场影响的粒子进行分离和回收的一种技术。利用该技术可以将纳米(或微米)粒子和生物微粒从水溶液中快速简便地分离出来。所以,磁分离技术被应用于生物工艺和生物医学领域,如:细胞排列、酶的固定、药物传输、蛋白分离等。该技术的优点是用最短的时间分离出最大量的目标物质。
在本论文中,我们制备了一系列基于N-卤胺的抗菌纳米复合材料,并研究了其对革兰氏阳性菌和革兰氏阴性菌的抗菌活性。另外,我们还引入了磁分离技术,为N-卤胺抗菌纳米复合材料的回收和分离提供了更加快速简便的方法。具体内容如下:
1.制备了N-卤胺功能化的SiO_2/聚苯乙烯核壳纳米粒子(SiO2@PS-N-卤胺),并研究了反应条件对纳米粒子形貌的影响。利用最小抑菌浓度法证明了SiO2@PS-N-卤胺纳米粒子对大肠杆菌和金黄色葡萄球菌都具有良好的抗菌效果,其抗菌活性为微米尺寸N-卤胺的2-8倍。SiO2@PS-N-卤胺纳米粒子对金黄色葡萄球菌的抗菌性能优于大肠杆菌。此外,研究还发现N-卤胺抗菌材料具有较高的稳定性。
2.制备了四种单分散的SiO_2@N-卤胺核壳纳米粒子,并对其结构、形貌及组成进行了表征。四种纳米粒子都具有规整的球形外貌和明显的核壳结构,且相互间没有太大差异。SiO2@N-卤胺纳米粒子的尺寸可以通过调节SiO_2模板粒子的大小来控制。利用滴定法对SiO_2@N-卤胺纳米粒子中的氯含量进行了定量测定。另外,通过抗菌实验研究了材料尺寸、氯含量、接触时间对SiO_2@N-卤胺纳米粒子的抗菌性能的影响。FTIR测试证明了SiO_2@N-卤胺纳米粒子的可再生性。杀菌后的SiO_2@N-卤胺纳米粒子经简单的氯化处理后,使其重新具有抗菌活性。
3.制备了单分散的Fe_3O_4@SiO2@N-卤胺纳米粒子,并利用多种测试手段对其进行了表征。通过抗菌实验研究了Fe_3O_4@SiO2@N-卤胺纳米粒子和微米尺寸的N-卤胺的抗菌性能。两种材料对金黄色葡萄球菌和绿脓杆菌都具有抑菌效果,且Fe_3O_4@SiO2@N-卤胺纳米粒子的抗菌效果明显优于微米尺寸的N-卤胺。通过抗菌动力学实验发现,Fe_3O_4@SiO2@N-卤胺纳米粒子的抗菌性随着材料氯含量的增加而随之增强。磁性能研究表明,Fe_3O_4@SiO2@N-卤胺纳米粒子具有超顺磁性,这种磁性特征可使该材料在水溶液中具有较好的可回收性。
4.制备了PSA@Fe_3O_4@SiO2-N-卤胺核壳纳米粒子,并研究了反应参数对纳米粒子形貌和各组分含量的影响。通过抗菌实验发现,PSA@Fe_3O_4@SiO2纳米粒子对金黄色葡萄球菌和绿脓杆菌没有抑菌效果,而微米尺寸的N-卤胺和PSA@Fe_3O_4@SiO2-N-卤胺纳米粒子对两种实验菌均具有抗菌效果,表明PSA@Fe_3O_4@SiO2-N-卤胺纳米粒子的抗菌性能源于N-卤胺。此外,N-卤胺抗菌材料对绿脓杆菌的抑菌效果优于金黄色葡萄球菌。磁性能研究表明,PSA@Fe_3O_4@SiO2-N-卤胺纳米粒子具有超顺磁性,且在水相中具有较强的磁响应性,可以通过外加磁场的方式对其进行分离和回收。
综上所述,本论文设计制备了多种基于N-卤胺的抗菌纳米复合材料,通过缩小材料尺寸有效提高了N-卤胺的抗菌活性。另外,通过引入磁分离技术,使N-卤胺抗菌材料的分离和回收变得更加简单快速。
In response to the wide spreading of infectious diseases caused by pathogen,antibacterial materials that can effectively inhibit the growth of microorganisms haveattracted significant research interests. To date, sodium hypochlorite, ozone, chlorineoxide, metal ions, quaternary ammonium salts, quaternary phosphonium salts,peptides, guanidinium, N-halamines, etc., have been used in the development ofantibacterial materials. Among them, N-halamine antibacterial materials have receivedintensive interest because of their unique properties, such as antibacterial efficacy,stability in aqueous solution and in dry storage, regenerability upon exposure towashing cycles, lack of corrosion, low toxicity, and relatively low expense. Due tothese characteristics, the application of N-halamine antibacterial materials rangesacross the area of medical devices, hospitals, water purification systems, foodpackaging, food storage, hygienic products, etc. The antibacterial mechanism ofN-halamine materials involves the direct transfer of halogen from the N-halamine tobacterial cells, and the halogen has a strong tendency to participate in ionic reactions,thereby leading to destruction or inhibition of metabolic processes in microorganisms.Antibacterial performances of N-halamine materials strongly depend on theiractivated surface area. N-halamine materials with larger surface area can provide moreN-halamine functional sites to contact with the bacteria, and the N-halamineincrement can lead to the enhanced antibacterial efficiency.
Nanomaterials with the size ranged from few nanometers to more than a hundrednanometers can present superior physical, chemical, and mechanical properties.Because of their small particle size and large surface area, nanomaterials possessremarkable potential and prospect. Therefore, to enhance antibacterial efficacy,fabrication of N-halamine materials with nanostructure to enlarge activate surface areais advisable. However, there is a major drawback to the application ofnanometer-sized N-halamine materials originating from the separation. Antibacterialperformance of N-halamine materials is usually conducted in an aqueous suspension,which requires an additional separation step to separate N-halamine nanomaterials from suspension, and separation of such fine nanostructures from a large volume ofsolution involves further expense.
The recent successful synthesis of magnetic nanoparticles provides a convenienttool for exploring magnetic separation technique due to their specific characteristics.Magnetic separation is a technology involving the transport of magnetic ormagnetically susceptible particles in a gradient magnetic field. This technology can beemployed to recover desired species of submicron dimensions from solutioncontaining suspended solid particles or other biological particulates in a rapid andeasy way. Therefore, magnetic separation has been widely applied to various aspectsin biotechnology and biomedicine, such as cell sorting, enzyme immobilization, drugdelivery, and protein separation. The advantage of magnetic separation technology isto maximize the amount of target particles removed from the suspension whileminimizing the amount of time to carry out the separation process.
In this thesis, a series of N-halamine-based antibacterial nanocomposite materialswere synthesized, and their antibacterial performances against Gram-positive andGram-negative bacteria were investigated. In addition, magnetic separation techniquewas introduced to facilitate the separation process of the N-halamine-basedantibacterial nanocomposite materials. The details are as follows:
1. N-halamine-functionalized SiO_2@PS core-shell nanoparticles(SiO_2@PS-N-halamine) were prepared, and the effect of reaction condition onmorphology was studied. The excellent antibacterial activity of SiO_2@PS-N-halaminenanoparticles against E. coli and S. aureus was assayed via the minimum inhibitionconcentration (MIC) method. Antibacterial test indicated that SiO_2@PS-N-halaminenanoparticles displayed2-8times higher biocidal activity than the micrometer-sizedN-halamine, and these powerful and stable nano-sized biocides had higherantibacterial efficacy against S. aureus than E. coli. Finally, the long-term stability ofN-halamine structural biocide was confirmed.
2. Four kinds of monodisperse SiO_2@N-halamine core-shell nanoparticles weresynthesized, and their structure, morphology, and component were characterizedsubsequently. All these nanoparticles have legible spherical shapes and obviouscore-shell structures, and no significant differences were observed in the morphologyamong them. Diameters of SiO_2@N-halamine nanoparticles were controlled viaadjusting the size of silica template. Chlorine contents of SiO_2@N-halamine nanoparticles were determined by the aid of iodometric/thiosulfate titration method.Effects of particle size, chlorine content, and contact time on antibacterial efficiencywere investigated. The regenerability feature of N-halamine-based materials wastestified by FTIR measurement. Hydantoin structure appeared after antibacterialperformance can convert to N-halamine via chlorination treatment.
3. Monodisperse Fe_3O_4@SiO_2@N-halamine nanoparticles were fabricated andcharacterized by a series of measurement. Antibacterial test revealed that both themicrometer-sized N-halamine and Fe_3O_4@SiO_2@N-halamine nanoparticles possessedantibacterial property, and Fe_3O_4@SiO_2@N-halamine nanoparticles showed higherantibacterial capacity than micrometer-sized counterpart. The antibacterial kinetic testconfirmed that the antibacterial efficiency of Fe_3O_4@SiO_2@N-halamine nanoparticlesincreased with chlorine content. Magnetism study revealed thatFe_3O_4@SiO_2@N-halamine nanoparticles have super-paramagnetic property, whichcan make them magnetically separable from the aquaeos solution.
4. PSA@Fe_3O_4@SiO_2-N-halamine core-shell nanoparticles were prepared, andeffects of reaction parameters on morphology and content were studied. Antibacterialtest revealed that both micrometer-sized N-halamine andPSA@Fe_3O_4@SiO_2-N-halamine nanoparticles have antibacterial activity against S.aureus and P. aeruginosa, while PSA@Fe_3O_4@SiO_2nanopartices without N-halaminemodification displayed no antibacterial property, indicating that antibacterialperformance of PSA@Fe_3O_4@SiO_2-N-halamine nanoparticles is provided by theN-halamine structure. Both these two N-halamine-structural materials have higherantibacterial efficacy against P. aeruginosa than S. aureus. Magnetism investigationshowed that PSA@Fe_3O_4@SiO_2-N-halamine nanoparticles have super-paramagneticproperty and magnetic response, and they can be separated readily by the aid of anexternal magnetic field.
In summary, several N-halamine-based antibacterial nanocomposite materialswere synthesized, and their antibacterial performances were enhanced effectivelythrough particle size decrease. In addition, magnetic separation technology wasintroduced to facilitate the separation process of N-halamine-based materials.
引文
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