醫(yī)用殼聚糖納米微球的制備及其性能研究
發(fā)布時間：2019-05-28 05:30
【摘要】：殼聚糖為一種陽離子聚合物，無毒性，無免疫原性，具有良好的生物降解性和生物相容性，在體內(nèi)可被溶菌酶等酶解成低聚糖。以殼聚糖分子為原料制備的殼聚糖納米微球是一種生物基因納米載體，可以結(jié)合、濃縮目的基因，，同時保護(hù)目的基因不受機(jī)體血漿或組織細(xì)胞中各種酶和補(bǔ)體的破壞，在基因治療方面具有良好的應(yīng)用前景。 目的：制備生物功能性的殼聚糖納米微球，探索納米微球制備過程中球粒徑的影響因素，對制備出的殼聚糖納米微球進(jìn)行化學(xué)表征，同時對其進(jìn)行生物學(xué)性能研究。方法：采用反相微乳液法制備殼聚糖納米微球，通過激光粒度測定儀、掃描電鏡、透射電鏡對其粒徑分布、Zeta電位分布和形貌特征進(jìn)行研究。通過細(xì)胞攝取實驗、MTT測試實驗和DNA包封率測定來評價制備出的殼聚糖納米微球的生物學(xué)性能。 結(jié)果：本實驗采用反相微乳液制備殼聚糖納米微球，殼聚糖溶液的濃度和pH值、水油相的體積比、表面活性劑、丙酮和反應(yīng)體系的溫度均可以影響納米微球的粒徑。在合適的反應(yīng)條件下，最終制備出的空白殼聚糖納米微球外形圓整，大小均一，分散性良好，粒徑分布為（179±12.85）nm，Zeta電勢分布為＋（31.4±5.87）mV；制備出的FITC@CS納米微球外形圓整，大小均一，分散性良好，粒徑分布為（363.2±94）nm，Zeta電勢分布為－（24.2±6.8）mV；制備出的pEGFP@CS納米微球外形圓整，大小均一，分散性良好，粒徑分布為（227.9±83.1）nm，Zeta電勢分布為＋（11.4±6.16）mV。MTT測試結(jié)果說明空白殼聚糖納米微球在一定濃度范圍內(nèi)對MC3T3細(xì)胞的增殖沒有明顯影響；共聚焦激光掃描生物顯微鏡觀察結(jié)果說明FITC標(biāo)記的殼聚糖納米微球可以被MC3T3細(xì)胞攝取入細(xì)胞內(nèi)；采用反相微乳液法能夠制備出攜帶pEGFP的殼聚糖納米微球，通過計算得出DNA的包封率為28.8%。 結(jié)論：在合適的條件下，采用反相微乳液法能夠制備出空白殼聚糖納米微球、FITC@CS納米微球、pEGFP@CS納米微球。最終制備出的殼聚糖納米微球毒性較低，生物相容性較好，可以被MC3T3-E1細(xì)胞攝取入胞內(nèi)，并且可以攜帶DNA，測得的DNA包封率為28.8%，是一個良好的生物納米基因載體。
[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 鹵5.87) mV;. The prepared FITC@CS nanoparticles have round shape, uniform size and good dispersibility. The particle size distribution is (363.2 鹵94) nm,Zeta potential distribution is-(24.2 鹵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 鹵83.1) nm,. The potential distribution of Zeta was + (11.4 鹵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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08
本文編號：2486775
[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 鹵5.87) mV;. The prepared FITC@CS nanoparticles have round shape, uniform size and good dispersibility. The particle size distribution is (363.2 鹵94) nm,Zeta potential distribution is-(24.2 鹵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 鹵83.1) nm,. The potential distribution of Zeta was + (11.4 鹵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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08
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