含糖溫敏聚合物納米纖維膜的制備及其性質(zhì)研究
發(fā)布時間：2018-09-05 05:53
【摘要】：近年來,基于納米級的尺寸和快速的溫度響應(yīng)性等特性,智能聚合物納米纖維作為新型智能高分子材料的研究在生物、醫(yī)藥等諸多領(lǐng)域備受關(guān)注。同時,由于糖基可以很大程度上改善高分子材料的親水性、生物相容性等性質(zhì),因此,含糖聚合物的合成研究也引起各國研究者的重視。 本論文在大量前人研究工作的基礎(chǔ)上,設(shè)計和制備了一系列糖衍生物和聚(N-異丙基丙烯酰胺)(PNIPAM)的新型熱敏性含糖聚合物,通過各種表征手段確認(rèn)其結(jié)構(gòu),并應(yīng)用靜電紡絲手段將相關(guān)聚合物制備成納米纖維,系統(tǒng)研究了基于其溫度敏感特性和含糖功能基團(tuán)的蛋白質(zhì)非特異性和特異性吸附性能研究,及其生物相容性的測定。主要研究內(nèi)容包括以下幾部分： 第一章,概述了溫度響應(yīng)性含糖高分子材料,基于PNIPAM的熱敏感性和含糖高分子材料的納米纖維膜的研究背景。 第二章,含糖溫敏性聚合物納米纖維膜的制備。為了研究不同的材料對于識別性能的影響,進(jìn)而研究特異性蛋白質(zhì)的識別機(jī)理,我們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),通過自由基聚合法將N-異丙基丙烯酰胺和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,制備出了一系列含糖溫敏共聚物Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、 Poly(NIPAM-co-OVEG)。利用核磁共振(1H NMR)和紅外光譜(FT-IR)對聚合物的結(jié)構(gòu)進(jìn)行了表征。利用可見光吸收法測定了Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)的低臨界溶解溫度(LCST)。結(jié)果顯示：采用自由基聚合制備的溫敏共聚物,較低臨界溶解溫度(LCST)由N-異丙基丙烯酰胺均聚物的32分別提高到了34、36、39℃((Poly(NIPAM-co-OVDG)其中NIPAM與OVDG的摩爾比分別為15：1、10：1、5：1)、34、35、36℃((Poly(NIPAM-co-OVZG)其中NIPAM與OVZG的摩爾比分別為20:1、15:1、7:1);36,38、39℃((Poly(NIPAM-co-OVEG)其中NIPAM與OVEG的摩爾比分別為7：1、15：1、20：1)。側(cè)鏈上的葡萄糖基還能對伴刀豆蛋白進(jìn)行特異性識別,在蛋白質(zhì)分離純化等領(lǐng)域有潛在的應(yīng)用價值。 應(yīng)用靜電紡絲的方法,將含糖溫敏共聚物Poly(NIPAM-co-OVDG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)和PLCL溶于二氯甲烷和無水乙醇混合溶液中,靜電紡制備含有含糖溫敏共聚物Poly(NIPAM-co-OVAG)、Poly(NIPAM-co-OVZG)、Poly(NIPAM-co-OVEG)的納米纖維。由于該纖維膜含有親水性極強(qiáng)的葡萄糖基,可以在水溶液中迅速溶解,因此通過與PLCL進(jìn)行共紡的方式降低納米纖維的溶解性。SEM表征結(jié)果看出,利用所制備的溫敏含糖聚合物通過靜電紡絲技術(shù)制備出了均勻的納米纖維膜,纖維直徑基本處于200-500nm。 第三章中,我們在第二章的基礎(chǔ)上,利用紫外分光光度計測定系列膜材料在不同的溫度下對蛋白質(zhì)的靜態(tài)吸附及其抗非特異性蛋白質(zhì)吸附的研究。在研究特異性蛋白質(zhì)的靜態(tài)吸附實(shí)驗(yàn)中,選擇Poly(NIPAM-co-OVDG)作為研究對象,研究不同溫度和不同膜材料對于特異性蛋白質(zhì)伴刀豆蛋白(Con A)的吸附。研究結(jié)果表明,在25和℃條件下,Con A的靜態(tài)吸附顯示出了差異。在Con A濃度為0.563mg/mL時,在37℃條件下,膜材料吸附的Con A量比在25℃條件下吸附量少,并且吸附速率也快,并且在不同的膜材料中,隨著膜材料中含糖量的增加,吸附的Con A量也在增多。但是當(dāng)Con A的濃度為0.1mg/mL的時候,吸附曲線在25和37℃條件下基本沒有什么區(qū)別。在本實(shí)驗(yàn)的基礎(chǔ)上,我們利用吸附公式計算出來不同溫度不同Con A濃度條件下,不同膜材料的膜表面吸附濃度。 在研究膜材料抗非特異性蛋白質(zhì)吸附的實(shí)驗(yàn)中,同時選擇Poly(NIPAM-co-OVDG)作為研究對象,研究不同溫度和不同膜材料對于非特異性蛋白質(zhì)牛血清白蛋白(BSA)的吸附。從結(jié)果中可以看出來,在相同的BSA濃度的條件下,膜材料在37℃條件下吸附量小于在25℃條件下 第四章中,我們在第二章的基礎(chǔ)上,研究不同的膜材料對特異性蛋白質(zhì)的吸附和解吸附測定。本實(shí)驗(yàn)中,我們選擇FITC標(biāo)記的Con A和RBITC標(biāo)記的BSA作為實(shí)驗(yàn)對象,研究不同的材料對于蛋白質(zhì)的吸附性能,通過Confocal來測定不同膜材料表面的熒光強(qiáng)度進(jìn)行定性分析,后又通過熒光分光光度計測定不同膜材料的熒光強(qiáng)度進(jìn)行定量分析。從實(shí)驗(yàn)結(jié)果可以看出來,本研究課題中合成的材料可以特異性的吸附FITC-Con A,而對RBITC-BSA卻沒有太多的吸附,這樣更加明顯的證明制備的系列膜材料可以很好的特異性吸附Con A的能力。并且以Poly(NIPAAm-co-OVDG)為研究對象,進(jìn)一步的研究了不同的溫度對于FITC-Con A的吸附影響,從實(shí)驗(yàn)結(jié)果中可以看出來,在37℃條件下,FITC-Con A的吸附強(qiáng)度要低于25℃條件下的吸附,與第三章中實(shí)驗(yàn)結(jié)果相符。 第五章中,我們在第二章的基礎(chǔ)上,研究不同膜材料的細(xì)胞毒性。本實(shí)驗(yàn)利用MTT測定方法,研究不同膜材料的生物相容性和細(xì)胞毒性。從實(shí)驗(yàn)結(jié)果可以看出來,不同的膜材料上細(xì)胞可以很好的存活,實(shí)驗(yàn)結(jié)果中吸光度遠(yuǎn)遠(yuǎn)高于對照組,證明制備的膜材料上細(xì)胞的存活率很高,膜材料具有很好的生物相容性。 我們研究了吸附有Con A的膜材料對于細(xì)胞增殖的影響。從實(shí)驗(yàn)結(jié)果中可以看出來,吸附有Con A的膜材料可以殺死HeLa細(xì)胞,從而為靶向釋藥的應(yīng)用研究提供了理論基礎(chǔ)。 第六章中,我們總結(jié)了本課題中研究的主要內(nèi)容,并對本課題的研究前景進(jìn)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.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：TQ340.64;TB383.2
本文編號：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.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：TQ340.64;TB383.2
【參考文獻(xiàn)】
相關(guān)博士學(xué)位論文 前2條
1 車愛馥;丙烯腈共聚物納米纖維膜的表面功能化及其識別性能研究[D];浙江大學(xué);2009年
2 胡夢欣;聚丙烯微孔膜的高密度糖基化及其應(yīng)用基礎(chǔ)研究[D];浙江大學(xué);2009年
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