含糖温敏聚合物纳米纤维膜的制备及其性质研究
发布时间：2018-09-05 05:53
【摘要】：近年来,基于纳米级的尺寸和快速的温度响应性等特性,智能聚合物纳米纤维作为新型智能高分子材料的研究在生物、医药等诸多领域备受关注。同时,由于糖基可以很大程度上改善高分子材料的亲水性、生物相容性等性质,因此,含糖聚合物的合成研究也引起各国研究者的重视。 本论文在大量前人研究工作的基础上,设计和制备了一系列糖衍生物和聚(N-异丙基丙烯酰胺)(PNIPAM)的新型热敏性含糖聚合物,通过各种表征手段确认其结构,并应用静电纺丝手段将相关聚合物制备成纳米纤维,系统研究了基于其温度敏感特性和含糖功能基团的蛋白质非特异性和特异性吸附性能研究,及其生物相容性的测定。主要研究内容包括以下几部分： 第一章,概述了温度响应性含糖高分子材料,基于PNIPAM的热敏感性和含糖高分子材料的纳米纤维膜的研究背景。 第二章,含糖温敏性聚合物纳米纤维膜的制备。为了研究不同的材料对于识别性能的影响,进而研究特异性蛋白质的识别机理,我们采用酶促法合成了可聚合的葡萄糖乙烯酯衍生物6-O-乙烯已二酰-D-葡萄糖(OVDG)、6-O-乙烯壬二酰-D-葡萄糖(OVZG)、6-O乙烯癸二酰-D-葡萄糖(OVEG),通过自由基聚合法将N-异丙基丙烯酰胺和6-O-乙烯已二酰-D-葡萄糖(OVDG)、6-O-乙烯壬二酰-D-葡萄糖(OVZG)、6-O-乙烯癸二酰-D-葡萄糖(OVEG)共聚,制备出了一系列含糖温敏共聚物Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、 Poly(NIPAM-co-OVEG)。利用核磁共振(1H NMR)和红外光谱(FT-IR)对聚合物的结构进行了表征。利用可见光吸收法测定了Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)的低临界溶解温度(LCST)。结果显示：采用自由基聚合制备的温敏共聚物,较低临界溶解温度(LCST)由N-异丙基丙烯酰胺均聚物的32分别提高到了34、36、39℃((Poly(NIPAM-co-OVDG)其中NIPAM与OVDG的摩尔比分别为15：1、10：1、5：1)、34、35、36℃((Poly(NIPAM-co-OVZG)其中NIPAM与OVZG的摩尔比分别为20:1、15:1、7:1);36,38、39℃((Poly(NIPAM-co-OVEG)其中NIPAM与OVEG的摩尔比分别为7：1、15：1、20：1)。侧链上的葡萄糖基还能对伴刀豆蛋白进行特异性识别,在蛋白质分离纯化等领域有潜在的应用价值。 应用静电纺丝的方法,将含糖温敏共聚物Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)和PLCL溶于二氯甲烷和无水乙醇混合溶液中,静电纺制备含有含糖温敏共聚物Poly(NIPAM-co-OVAG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)的纳米纤维。由于该纤维膜含有亲水性极强的葡萄糖基,可以在水溶液中迅速溶解,因此通过与PLCL进行共纺的方式降低纳米纤维的溶解性。SEM表征结果看出,利用所制备的温敏含糖聚合物通过静电纺丝技术制备出了均匀的纳米纤维膜,纤维直径基本处于200-500nm。 第三章中,我们在第二章的基础上,利用紫外分光光度计测定系列膜材料在不同的温度下对蛋白质的静态吸附及其抗非特异性蛋白质吸附的研究。在研究特异性蛋白质的静态吸附实验中,选择Poly(NIPAM-co-OVDG)作为研究对象,研究不同温度和不同膜材料对于特异性蛋白质伴刀豆蛋白(Con A)的吸附。研究结果表明,在25和℃条件下,Con A的静态吸附显示出了差异。在Con A浓度为0.563mg/mL时,在37℃条件下,膜材料吸附的Con A量比在25℃条件下吸附量少,并且吸附速率也快,并且在不同的膜材料中,随着膜材料中含糖量的增加,吸附的Con A量也在增多。但是当Con A的浓度为0.1mg/mL的时候,吸附曲线在25和37℃条件下基本没有什么区别。在本实验的基础上,我们利用吸附公式计算出来不同温度不同Con A浓度条件下,不同膜材料的膜表面吸附浓度。 在研究膜材料抗非特异性蛋白质吸附的实验中,同时选择Poly(NIPAM-co-OVDG)作为研究对象,研究不同温度和不同膜材料对于非特异性蛋白质牛血清白蛋白(BSA)的吸附。从结果中可以看出来,在相同的BSA浓度的条件下,膜材料在37℃条件下吸附量小于在25℃条件下 第四章中,我们在第二章的基础上,研究不同的膜材料对特异性蛋白质的吸附和解吸附测定。本实验中,我们选择FITC标记的Con A和RBITC标记的BSA作为实验对象,研究不同的材料对于蛋白质的吸附性能,通过Confocal来测定不同膜材料表面的荧光强度进行定性分析,后又通过荧光分光光度计测定不同膜材料的荧光强度进行定量分析。从实验结果可以看出来,本研究课题中合成的材料可以特异性的吸附FITC-Con A,而对RBITC-BSA却没有太多的吸附,这样更加明显的证明制备的系列膜材料可以很好的特异性吸附Con A的能力。并且以Poly(NIPAAm-co-OVDG)为研究对象,进一步的研究了不同的温度对于FITC-Con A的吸附影响,从实验结果中可以看出来,在37℃条件下,FITC-Con A的吸附强度要低于25℃条件下的吸附,与第三章中实验结果相符。 第五章中,我们在第二章的基础上,研究不同膜材料的细胞毒性。本实验利用MTT测定方法,研究不同膜材料的生物相容性和细胞毒性。从实验结果可以看出来,不同的膜材料上细胞可以很好的存活,实验结果中吸光度远远高于对照组,证明制备的膜材料上细胞的存活率很高,膜材料具有很好的生物相容性。 我们研究了吸附有Con A的膜材料对于细胞增殖的影响。从实验结果中可以看出来,吸附有Con A的膜材料可以杀死HeLa细胞,从而为靶向释药的应用研究提供了理论基础。 第六章中,我们总结了本课题中研究的主要内容,并对本课题的研究前景进行了展望。
[Abstract]:In recent years, intelligent polymer nanofibers have attracted much attention in many fields, such as biology, medicine and so on, because of their nano-scale size and fast temperature response. The synthesis of complexes has attracted the attention of researchers all over the world.
In this paper, a series of novel thermosensitive polysaccharides containing polysaccharides derivatives and poly (N-isopropylacrylamide) (PNIPAM) were designed and synthesized on the basis of a large number of previous studies. Their structures were confirmed by various characterization methods. The related polymers were prepared into nanofibers by electrospinning, and their temperature-based properties were systematically studied. Sensitivity, nonspecific and specific adsorption properties of proteins containing sugary functional groups, and determination of their biocompatibility were studied.
In the first chapter, the research background of temperature-responsive sugary polymer materials, PNIPAM-based thermal sensitivity and sugary polymer nanofiber membranes is summarized.
In order to study the effect of different materials on the recognition performance and the recognition mechanism of specific proteins, we synthesized polymerizable glucose ethylene ester derivatives 6-O-vinylhexanedioyl-D-glucose (OVDG) and 6-O-vinylazelaoyl-D-glucose (6-O-vinylazelaoyl-D-glucose) by enzymatic method. OVZG, 6-O-vinyl sebacoyl-D-glucose (OVEG), N-isopropyl acrylamide, 6-O-vinylhexanedioyl-D-glucose (OVDG), 6-O-vinylnonyldiacyl-D-glucose (OVZG), 6-O-vinyl sebacoyl-D-glucose (OVEG) were copolymerized by free radical polymerization to prepare a series of polysaccharide-containing thermosensitive copolymers (PAM-co-OVDG), poly (PANIZM-co-OVDG), poly (PAVZG). Poly (NIPAM-co-OVEG). The structure of the polymer was characterized by 1H NMR and FT-IR. The low critical solution temperature (LCST) of poly (NIPAM-co-OVDG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG) were determined by visible light absorption method. The LCST of N-isopropylacrylamide homopolymer was increased from 32 to 34,36,39 ((Poly (NIPAM-co-OVDG), in which the molar ratios of NIPAM to OVDG were 15:1,10:1,5:1), 34,35,36 ((Poly (NIPAM-co-OVZG), in which the molar ratios of NIPAM to OVZG were 20:1,15:1,7:1) and 36,38,39 ((Poly (PANIPAM-co-OVEG), respectively). The molar ratios of M to OVEG were 7:1,15:1,20:1, respectively. Glucosyl groups on the side chains could also recognize concanavalin specifically, which could be used in protein isolation and purification.
Poly (NIPAM-co-OVDG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG) were dissolved in a mixture of dichloromethane and anhydrous ethanol by electrospinning to prepare nanofibers containing poly (NIPAM-co-OVAG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG). The membrane contains highly hydrophilic glucose group and can dissolve rapidly in aqueous solution. Therefore, the solubility of nanofibers can be reduced by co-spinning with PLCL. SEM characterization results show that uniform nanofibers were prepared by electrospinning using the prepared thermo-sensitive polysaccharide polymer. The diameter of nanofibers is basically 200-500n. M.
In the third chapter, on the basis of the second chapter, we used ultraviolet spectrophotometer to measure the static adsorption of proteins and the anti-nonspecific adsorption of proteins by a series of membrane materials at different temperatures. The results showed that the static adsorption of Con A was different at 25 and C. At the concentration of Con A 0.563 mg/mL, at 37 C, the amount of Con A adsorbed by the membrane material was less than that at 25 C, and the adsorption rate was faster. However, when the concentration of Con A was 0.1mg/mL, the adsorption curves were almost the same at 25 and 37 C. On the basis of this experiment, we calculated the adsorption formula at different temperatures and different Con A concentrations, and the adsorption curves were different. Membrane surface adsorption concentration of membrane materials.
Poly (NIPAM-co-OVDG) was selected as the research object in the experiment of anti-nonspecific protein adsorption of membrane materials. The adsorption of nonspecific protein bovine serum albumin (BSA) on the membrane materials at different temperatures and different membrane materials was studied. It can be seen from the results that under the same BSA concentration, the membrane materials adsorbed at 37 C. The attachment is less than 25 C.
In Chapter 4, we studied the adsorption and desorption of specific proteins on different membrane materials on the basis of Chapter 2. In this experiment, we selected FITC-labeled ConA and RBITC-labeled BSA as the experimental objects, studied the adsorption properties of different materials for proteins, and measured the surface of different membrane materials by Confocus. The fluorescence intensity was qualitatively analyzed, and then the fluorescence intensity of different membrane materials was measured by fluorescence spectrophotometer for quantitative analysis. From the experimental results, we can see that the materials synthesized in this study can specifically adsorb FITC-Con A, but there is not much adsorption on RBITC-BSA, so it is more obvious to prove the prepared system. Poly (NIPAAm-co-OVDG) was used as the research object to further study the effect of different temperatures on the adsorption of FITC-Con A. From the experimental results, it can be seen that the adsorption strength of FITC-Con A at 37 C is lower than that at 25 C, which is confirmed in Chapter 3. The test results are consistent.
In the fifth chapter, we studied the cytotoxicity of different membrane materials on the basis of the second chapter. In this experiment, we used MTT assay to study the biocompatibility and cytotoxicity of different membrane materials. The cell survival rate of the prepared membrane material is very high, and the membrane material has good biocompatibility.
We studied the effect of membrane materials adsorbed with Con A on the proliferation of HeLa cells. It can be seen from the experimental results that the membrane materials adsorbed with Con A can kill HeLa cells, thus providing a theoretical basis for the application of targeted drug release.
In the sixth chapter, we summarize the main contents of this subject and look forward to the future of this subject.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TQ340.64;TB383.2
本文编号：2223393
[Abstract]:In recent years, intelligent polymer nanofibers have attracted much attention in many fields, such as biology, medicine and so on, because of their nano-scale size and fast temperature response. The synthesis of complexes has attracted the attention of researchers all over the world.
In this paper, a series of novel thermosensitive polysaccharides containing polysaccharides derivatives and poly (N-isopropylacrylamide) (PNIPAM) were designed and synthesized on the basis of a large number of previous studies. Their structures were confirmed by various characterization methods. The related polymers were prepared into nanofibers by electrospinning, and their temperature-based properties were systematically studied. Sensitivity, nonspecific and specific adsorption properties of proteins containing sugary functional groups, and determination of their biocompatibility were studied.
In the first chapter, the research background of temperature-responsive sugary polymer materials, PNIPAM-based thermal sensitivity and sugary polymer nanofiber membranes is summarized.
In order to study the effect of different materials on the recognition performance and the recognition mechanism of specific proteins, we synthesized polymerizable glucose ethylene ester derivatives 6-O-vinylhexanedioyl-D-glucose (OVDG) and 6-O-vinylazelaoyl-D-glucose (6-O-vinylazelaoyl-D-glucose) by enzymatic method. OVZG, 6-O-vinyl sebacoyl-D-glucose (OVEG), N-isopropyl acrylamide, 6-O-vinylhexanedioyl-D-glucose (OVDG), 6-O-vinylnonyldiacyl-D-glucose (OVZG), 6-O-vinyl sebacoyl-D-glucose (OVEG) were copolymerized by free radical polymerization to prepare a series of polysaccharide-containing thermosensitive copolymers (PAM-co-OVDG), poly (PANIZM-co-OVDG), poly (PAVZG). Poly (NIPAM-co-OVEG). The structure of the polymer was characterized by 1H NMR and FT-IR. The low critical solution temperature (LCST) of poly (NIPAM-co-OVDG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG) were determined by visible light absorption method. The LCST of N-isopropylacrylamide homopolymer was increased from 32 to 34,36,39 ((Poly (NIPAM-co-OVDG), in which the molar ratios of NIPAM to OVDG were 15:1,10:1,5:1), 34,35,36 ((Poly (NIPAM-co-OVZG), in which the molar ratios of NIPAM to OVZG were 20:1,15:1,7:1) and 36,38,39 ((Poly (PANIPAM-co-OVEG), respectively). The molar ratios of M to OVEG were 7:1,15:1,20:1, respectively. Glucosyl groups on the side chains could also recognize concanavalin specifically, which could be used in protein isolation and purification.
Poly (NIPAM-co-OVDG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG) were dissolved in a mixture of dichloromethane and anhydrous ethanol by electrospinning to prepare nanofibers containing poly (NIPAM-co-OVAG), poly (NIPAM-co-OVZG), poly (NIPAM-co-OVEG) and poly (NIPAM-co-OVEG). The membrane contains highly hydrophilic glucose group and can dissolve rapidly in aqueous solution. Therefore, the solubility of nanofibers can be reduced by co-spinning with PLCL. SEM characterization results show that uniform nanofibers were prepared by electrospinning using the prepared thermo-sensitive polysaccharide polymer. The diameter of nanofibers is basically 200-500n. M.
In the third chapter, on the basis of the second chapter, we used ultraviolet spectrophotometer to measure the static adsorption of proteins and the anti-nonspecific adsorption of proteins by a series of membrane materials at different temperatures. The results showed that the static adsorption of Con A was different at 25 and C. At the concentration of Con A 0.563 mg/mL, at 37 C, the amount of Con A adsorbed by the membrane material was less than that at 25 C, and the adsorption rate was faster. However, when the concentration of Con A was 0.1mg/mL, the adsorption curves were almost the same at 25 and 37 C. On the basis of this experiment, we calculated the adsorption formula at different temperatures and different Con A concentrations, and the adsorption curves were different. Membrane surface adsorption concentration of membrane materials.
Poly (NIPAM-co-OVDG) was selected as the research object in the experiment of anti-nonspecific protein adsorption of membrane materials. The adsorption of nonspecific protein bovine serum albumin (BSA) on the membrane materials at different temperatures and different membrane materials was studied. It can be seen from the results that under the same BSA concentration, the membrane materials adsorbed at 37 C. The attachment is less than 25 C.
In Chapter 4, we studied the adsorption and desorption of specific proteins on different membrane materials on the basis of Chapter 2. In this experiment, we selected FITC-labeled ConA and RBITC-labeled BSA as the experimental objects, studied the adsorption properties of different materials for proteins, and measured the surface of different membrane materials by Confocus. The fluorescence intensity was qualitatively analyzed, and then the fluorescence intensity of different membrane materials was measured by fluorescence spectrophotometer for quantitative analysis. From the experimental results, we can see that the materials synthesized in this study can specifically adsorb FITC-Con A, but there is not much adsorption on RBITC-BSA, so it is more obvious to prove the prepared system. Poly (NIPAAm-co-OVDG) was used as the research object to further study the effect of different temperatures on the adsorption of FITC-Con A. From the experimental results, it can be seen that the adsorption strength of FITC-Con A at 37 C is lower than that at 25 C, which is confirmed in Chapter 3. The test results are consistent.
In the fifth chapter, we studied the cytotoxicity of different membrane materials on the basis of the second chapter. In this experiment, we used MTT assay to study the biocompatibility and cytotoxicity of different membrane materials. The cell survival rate of the prepared membrane material is very high, and the membrane material has good biocompatibility.
We studied the effect of membrane materials adsorbed with Con A on the proliferation of HeLa cells. It can be seen from the experimental results that the membrane materials adsorbed with Con A can kill HeLa cells, thus providing a theoretical basis for the application of targeted drug release.
In the sixth chapter, we summarize the main contents of this subject and look forward to the future of this subject.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TQ340.64;TB383.2
【参考文献】
相关博士学位论文 前2条
1 车爱馥;丙烯腈共聚物纳米纤维膜的表面功能化及其识别性能研究[D];浙江大学;2009年
2 胡梦欣;聚丙烯微孔膜的高密度糖基化及其应用基础研究[D];浙江大学;2009年
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