医用壳聚糖纳米微球的制备及其性能研究

发布时间：2019-05-28 05:30

【摘要】：壳聚糖为一种阳离子聚合物，无毒性，无免疫原性，具有良好的生物降解性和生物相容性，在体内可被溶菌酶等酶解成低聚糖。以壳聚糖分子为原料制备的壳聚糖纳米微球是一种生物基因纳米载体，可以结合、浓缩目的基因，，同时保护目的基因不受机体血浆或组织细胞中各种酶和补体的破坏，在基因治疗方面具有良好的应用前景。目的：制备生物功能性的壳聚糖纳米微球，探索纳米微球制备过程中球粒径的影响因素，对制备出的壳聚糖纳米微球进行化学表征，同时对其进行生物学性能研究。方法：采用反相微乳液法制备壳聚糖纳米微球，通过激光粒度测定仪、扫描电镜、透射电镜对其粒径分布、Zeta电位分布和形貌特征进行研究。通过细胞摄取实验、MTT测试实验和DNA包封率测定来评价制备出的壳聚糖纳米微球的生物学性能。结果：本实验采用反相微乳液制备壳聚糖纳米微球，壳聚糖溶液的浓度和pH值、水油相的体积比、表面活性剂、丙酮和反应体系的温度均可以影响纳米微球的粒径。在合适的反应条件下，最终制备出的空白壳聚糖纳米微球外形圆整，大小均一，分散性良好，粒径分布为（179±12.85）nm，Zeta电势分布为＋（31.4±5.87）mV；制备出的FITC@CS纳米微球外形圆整，大小均一，分散性良好，粒径分布为（363.2±94）nm，Zeta电势分布为－（24.2±6.8）mV；制备出的pEGFP@CS纳米微球外形圆整，大小均一，分散性良好，粒径分布为（227.9±83.1）nm，Zeta电势分布为＋（11.4±6.16）mV。MTT测试结果说明空白壳聚糖纳米微球在一定浓度范围内对MC3T3细胞的增殖没有明显影响；共聚焦激光扫描生物显微镜观察结果说明FITC标记的壳聚糖纳米微球可以被MC3T3细胞摄取入细胞内；采用反相微乳液法能够制备出携带pEGFP的壳聚糖纳米微球，通过计算得出DNA的包封率为28.8%。结论：在合适的条件下，采用反相微乳液法能够制备出空白壳聚糖纳米微球、FITC@CS纳米微球、pEGFP@CS纳米微球。最终制备出的壳聚糖纳米微球毒性较低，生物相容性较好，可以被MC3T3-E1细胞摄取入胞内，并且可以携带DNA，测得的DNA包封率为28.8%，是一个良好的生物纳米基因载体。
[Abstract]:Chitosan is a kind of cationic polymer, which has no toxicity, no immunogenicity, good biodegradability and biocompatibility, and can be hydrolyzed into oligosaccharide by lysozyme and other enzymes in vivo. Chitosan nanoparticles prepared from chitosan molecules are a kind of biological gene nanocarrier, which can bind and concentrate the target gene, and protect the target gene from the destruction of various enzymes and complements in plasma or tissue cells. It has a good application prospect in gene therapy. Aim: to prepare biofunctional chitosan nanoparticles, to explore the factors affecting the particle size of chitosan nanoparticles, to characterize the prepared chitosan nanoparticles and to study their biological properties. Methods: chitosan nanoparticles were prepared by inverse microemulsion method. The particle size distribution, Zeta potential distribution and morphology of chitosan nanoparticles were studied by laser particle size tester, scanning electron microscope and transmission electron microscope. The biological properties of chitosan nanoparticles were evaluated by cell uptake test, MTT test and DNA entrapment efficiency test. Results: chitosan nanoparticles were prepared by inverse microemulsion. The concentration and pH value of chitosan solution, the volume ratio of water to oil phase, surfactants, acetone and the temperature of reaction system could affect the particle size of chitosan nanoparticles. Under suitable reaction conditions, the blank chitosan nanoparticles prepared were round in shape, uniform in size and good in dispersion. the particle size distribution was (17912.85) nm,Zeta potential distribution was + (31.4 卤5.87) mV;. The prepared FITC@CS nanoparticles have round shape, uniform size and good dispersibility. The particle size distribution is (363.2 卤94) nm,Zeta potential distribution is-(24.2 卤6.8) mV;. The prepared pEGFP@CS nanoparticles are round in shape, uniform in size and good in dispersibility. The particle size distribution of the microspheres is (227.9 卤83.1) nm,. The potential distribution of Zeta was + (11.4 卤6.16) mV.MTT. The results showed that the blank chitosan nanoparticles had no significant effect on the proliferation of MC3T3 cells in a certain concentration range. The results of confocal laser scanning biomicroscopy showed that FITC-labeled chitosan nanoparticles could be ingested into MC3T3 cells. Chitosan nanoparticles carrying pEGFP can be prepared by inverse microemulsion method. The encapsulation efficiency of DNA is 28.8%. Conclusion: under suitable conditions, blank chitosan nanoparticles, FITC@CS nanoparticles and pEGFP@CS nanoparticles can be prepared by inverse microemulsion method. The chitosan nanoparticles prepared have low toxicity and good biocompatibility. They can be absorbed into the cells by MC3T3-E1 cells, and the DNA entrapment efficiency measured by DNA, is 28.8%. It is a good biological nano-gene vector.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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