组氨酸改性壳聚糖材料在仿生合成和蛋白分离的应用研究

发布时间：2018-04-12 12:41

本文选题：N-组氨酸壳聚糖 + 聚乳酸　；参考：《华侨大学》2014年博士论文

【摘要】：壳聚糖和聚乳酸是两种性能优良的生物材料，在组织工程和药物释放的应用上均显示其优越性。组氨酸具有很好的pH响应性，将组氨酸接枝到壳聚糖上制备N-组氨酸壳聚糖（NHCS），N-组氨酸壳聚糖保留了壳聚糖上的羟基和部分未取代的氨基以及新增加的具有pH响应性的咪唑基团，，保证了其具有良好的生物活性和pH响应性，以期获得一种综合性能优异的“复合型”组织工程材料（N-组氨酸壳聚糖支架、N-组氨酸壳聚糖/聚乳酸复合支架），可以进一步拓宽壳聚糖、聚乳酸在生物医学材料的应用。本文主要研究了组氨酸改性壳聚糖及其与聚乳酸复合材料的制备，并着重研究这些新材料在仿生合成和蛋白分离上的应用。本文利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺将组氨酸接枝到壳聚糖上，利用冷冻干燥法和浓缩干燥法分别制备N-组氨酸壳聚糖多孔支架（Ns）和N-组氨酸壳聚糖粉末（Np）；通过改变组氨酸/壳聚糖的摩尔比及壳聚糖分子量制得一系列不同取代度的N-组氨酸壳聚糖多孔支架（Ns）和N-组氨酸壳聚糖粉末（Np），通过FT-IR、1H NMR、EA、XRD、TGA和SEM等来表征，结果表明NHCS的取代度在3~11%之间，并且取代度受组氨酸的投加量和壳聚糖分子量的影响，组氨酸的投加量增大或壳聚糖分子量减小，取代度提高，支架孔径增大。NHCS均可形成泡沫状、海绵状等多种多孔材料，孔尺寸在5~120μm之间，孔隙率均大于85%，NHCS支架可望满足成纤维细胞、皮肤组织重建和骨组织工程的需要。在冷冻诱导相分离制备N-组氨酸壳聚糖多孔支架（Ns）的基础上进行二次相分离，考察不同取代度的N-组氨酸壳聚糖多孔支架和不同的N-组氨酸壳聚糖/聚乳酸质量之比等因素，制备一系列N-组氨酸壳聚糖/聚乳酸复合（NHCS/PLLA）支架，通过FT-IR、XRD、TGA和SEM等来表征N-组氨酸壳聚糖/聚乳酸复合支架，结果表明，NHCS/PLLA质量之比减小，复合支架孔隙率减小，密度增大。复合支架材料的孔尺寸约在13~18μm，孔隙率均大于92%，不同组成的NHCS/PLLA支架的力学性能测试表明其抗压强度和弹性模量分别在0.33~0.78MPa和1.75~5.28MPa之间，有望适用于软骨组织工程支架。利用浓缩干燥法所制备N-组氨酸壳聚糖粉末（Np）作为有机基质，在模拟体液中仿生调控合成纳米羟基磷灰石，探讨组氨酸/壳聚糖摩尔比、壳聚糖分子量、钙离子初始浓度、温度和陈化时间等因素对NHCS调控合成羟基磷灰石的影响，并以不添加NHCS的水体系、模拟体液体系作对照，研究结果表明在不添加NHCS的水体系、模拟体液体系都能得到微米级的羟基磷灰石块体。但在添加NHCS的模拟体液体系中可有效控制合成球形和梭状的纳米羟基磷灰石，以壳聚糖分子量为50kD、组氨酸与壳聚糖摩尔比为2:1所制备的NHCS（Np7）为有机基质，NHCS投加量为0.01g、Ca2+离子初始浓度为0.01mol·L-1、反应温度为37.0℃、陈化时间为24h时调控效果最好，梭状HAP长~200nm，宽~40nm，且含有少量碳羟基磷灰石，与自然骨成分相似。本研究还对NHCS不同投加量影响羟基磷灰石形成的机理作初步探讨，为仿生合成羟基磷灰石及其骨骼修复等提供参考。利用浓缩干燥法所制备N-组氨酸壳聚糖粉末（Np）作为有机基质，在水体系中仿生调控合成碳酸钙，探讨不同的组氨酸/壳聚糖摩尔比、壳聚糖分子量、钙离子初始浓度、pH值、温度和陈化时间等因素对NHCS调控合成碳酸钙的影响，并以不添加NHCS的水体系作对照。结果表明，在纯水条件下，生成的产品为颗粒较大单一立方体的方解石。NHCS的添加能对合成的碳酸钙晶型及形貌起到调控作用。NHCS在仿生合成碳酸钙过程对体系pH具有响应作用，体系pH不同，球形球霰石晶体的含量也不同，当pH为6.5时，球霰石的含量最低为66.2%，当pH远离6.5时，球霰石的含量可增加到93.7%。当陈化时间的延长，球霰石的含量缓慢减小，24h后球霰石的含量为86.2%。此外，对体系pH值、陈化时间等因素影响球霰石形成的机理也作初步探讨，为仿生合成其他生物矿物提供借鉴。探究NHCS支架、NHCS/PLLA复合支架的吸附牛血清白蛋白（BSA）的性能，研究结果表明NHCS支架、NHCS/PLLA复合支架对牛血清白蛋白溶液吸附效果良好，吸附容量Qe在335.84~1048.64mg·g-1之间。单纯NHCS支架中Ns10对BSA溶液的吸附能力最好，吸附容量Qe达820.90mg·g-1，而复合支架种以NPs3较佳，吸附容量Qe高达928.53mg·g-1。Ns10和NPs3可重复使用，重复吸附洗脱五次后的两种支架对BSA的吸附容量只降低了1.00%左右，有望为BSA或其他蛋白的分离纯化及回收利用提供新的载体，也有望作为组织工程用的支架材料。
[Abstract]:Chitosan and polylactide are two kinds of biological materials with excellent performance, showed its superiority in the application of tissue engineering and drug release. Histidine has good pH response, the histidine was grafted to chitosan preparation N- histidine chitosan (NHCS), N- histidine retained chitosan chitosan the hydroxyl and partially substituted amino and imidazole groups increased with pH response of, ensure its good biological activity and pH response, in order to obtain a kind of excellent performance composite materials for tissue engineering (N- histidine N- histidine chitosan scaffold, chitosan / polylactic acid composite), stent can further broaden chitosan, application of polylactic acid in biomedical materials. This paper mainly studies the histidine modified chitosan and polylactic acid composite material preparation, and focuses on the new material in bionic joint The application of the separation between the formation and the protein.
This paper uses 1- (3- two dimethylamino propyl) ethyl -3- carbon two imine hydrochloride and N- hydroxysuccinimide histidine grafted onto chitosan were prepared, N- histidine chitosan porous scaffolds by freeze-drying method and drying method (Ns) and N- histidine chitosan powder (Np); the change of histidine / chitosan the molar ratio and molecular weight of chitosan to prepare a series of different substituted N- histidine porous chitosan scaffolds of N- (Ns) and histidine chitosan powder (Np), by FT-IR, 1H NMR, EA, XRD, TGA and SEM to characterize the results show that the degree of substitution of NHCS in 3~11%, and the degree of substitution the histidine dosage and molecular weight of chitosan, the dosage of chitosan molecular weight increases or decreases the degree of substitution of histidine, improve stent diameter increases.NHCS can form a foam, sponge and other porous materials, pore size in 5~120 M The porosity is greater than 85%. NHCS scaffolds are expected to meet the needs of fibroblasts, skin tissue reconstruction and bone tissue engineering.
In the preparation of N- histidine chitosan porous scaffolds in frozen phase separation (Ns) on the basis of the two phase separation, the effects of different N- histidine chitosan porous scaffolds and the degree of substitution of histidine N- chitosan / poly lactic acid of different factors than the quality, the preparation of a series of N- histidine chitosan / polylactic acid composite (NHCS/PLLA) support, through FT-IR, XRD, TGA and SEM are used to characterize N- histidine chitosan / polylactic acid composite scaffolds, the results show that the NHCS/PLLA mass ratio decreases, the scaffold porosity decreases and the density increases. The pore size of composite scaffold materials in about 13~18 m, the porosity is greater than 92%, the mechanical performance test of NHCS/PLLA support different composition show that the compressive strength and elastic modulus were between 0.33~0.78MPa and 1.75~5.28MPa, is expected to apply to the scaffold for cartilage tissue engineering.
By using concentrated drying method prepared N- histidine chitosan powder (Np) was used as the organic matrix in the simulated body fluid regulation of biomimetic preparation of nano hydroxyapatite / chitosan on histidine, molar ratio, molecular weight of chitosan and calcium ion concentration, temperature and aging time and other factors on the synthesis of hydroxyapatite in the regulation of NHCS, and the do not add NHCS water system, simulated body fluid system as control. The results show that in NHCS was not added to the water system can be simulated body fluid system block hydroxyapatite micron. Nano hydroxyapatite in simulated body fluid, but the addition of NHCS can effectively control the synthesis of spherical and spindle shaped, with molecular weight of chitosan 50kD, histidine and chitosan molar ratio of 2:1 prepared by NHCS (Np7) as the organic matrix, the NHCS dosage of 0.01g, initial concentration of Ca2+ ions was 0.01mol L-1, the reaction temperature is 37 DEG C, Chen Time is the best control effect 24h, Clostridium HAP long ~200nm, wide ~40nm, which contains a small amount of carbon hydroxyapatite, similar to natural bone composition. The study of different NHCS dosage effect of hydroxyapatite formation mechanism are discussed, to provide reference for the synthesis of hydroxyapatite and its bionic bone repair.
By using concentrated drying method prepared N- histidine chitosan powder (Np) was used as the organic matrix in water system regulation biomimetic synthesis of calcium carbonate, explore different histidine / chitosan ratio, molecular weight of chitosan and calcium ion concentration, pH value, temperature and aging time and other factors on the regulation of NHCS carbonate synthesis calcium, and to add NHCS water system as control. The results showed that in pure water under the condition of generating products to add.NHCS calcite particles larger the single cube in biomimetic synthesis of calcium carbonate has effect on the pH response system of calcium carbonate crystal form and morphology play a regulatory role of.NHCS system. PH, the content of spherical vaterite crystals are different, when pH is 6.5, the lowest content of vaterite was 66.2%, when the pH is far from 6.5, the content of vaterite can be increased to 93.7%. when the aging time prolonged, the content of vaterite The content of 24h decreases slowly, vaterite is 86.2%. in addition, the system pH value, formation mechanism of vaterite aging time and other factors are discussed, to provide reference for the synthesis of other mineral biological bionic.
Study on NHCS NHCS/PLLA scaffolds, composite scaffolds for adsorption of bovine serum albumin (BSA) performance, the results show that the NHCS composite scaffold bracket, effect of NHCS/PLLA on bovine serum albumin adsorption, adsorption capacity of Qe between 335.84~1048.64mg g-1. NHCS Ns10 BSA to support simple solution adsorption ability best adsorption capacity of Qe reached 820.90mg - g-1 the composite scaffold with NPs3, better adsorption capacity of Qe reached 928.53mg - g-1.Ns10 and NPs3 can be used repeatedly, repeated adsorption and desorption adsorption capacity of two kinds of stents after five times of BSA was reduced by only 1% the left and right, purification and recycling is expected to provide a new carrier separation BSA or other protein, also is expected as the scaffold of tissue engineering.

【学位授予单位】：华侨大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08

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