透明质酸粘多糖的分子表征、流变学性质及其物理凝胶的研究
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摘要
粘多糖是广泛存在于动物体内的一类多糖,动物体内的多糖除了作为能量代谢的糖元外,基本上都属于粘多糖。透明质酸是粘多糖中最具代表性的一种,因为透明质酸被认为是唯一几乎存在于从细菌到人类所有动物体之中的粘多糖。透明质酸具有多样的生理功能和优良的物化性质,同时也是我国卫生部公布的第一批新资源食品之一,已被广泛应用于医药、食品、化妆品等领域。随着对透明质酸研究的深入,透明质酸在组织工程、纳米材料等领域也显示出了巨大的应用潜力。
透明质酸由葡萄糖醛酸和乙酰氨基葡萄糖双糖重复单元组成,其分子量可高达上百万,透明质酸的生理功能和应用特性与其分子量、分子量分布等分子参数密切相关;此外,粘弹性也是透明质酸重要的特性之一,透明质酸在体内所发挥的维持组织形态、润滑关节和缓冲应力等作用均与其粘弹性密切相关;凝胶化作用是一些多糖大分子最主要的特性之一,在制备透明质酸凝胶时如何保持其天然的生物相容性同时提高其应用的安全性是相关研究的焦点问题。本论文利用多种技术手段对透明质酸的分子参数进行了详细的表征,并对透明质酸的流变学性质及其冷冻解冻物理凝胶进行了系统的研究,主要内容及结论如下:
对一系列透明质酸样品的分子参数(分子量、分子量分布、分子尺寸及分子构象等)的多手段表征进行了系统性的对比研究。结果显示,对于Mw低于约2×106的样品来说,粘度法、CG-MALLS、SEC-MALLS、AFlFFF-MALLS和AGE法均能给出准确的表征结果,表征结果所存在的略微差异是由于过滤过程应用于不同的表征手段。对于Mw高于约2×106的样品来说,粘度法、CG-MALLS、SEC-MALLS、AFlFFF-MALLS给出了低于AGE法的Mw表征结果,这是由于透明质酸溶液中存在大尺寸的分子缔合或聚集所引起的,而这种分子聚集体无法用粘度法和静态光散射法进行准确的表征,但是可以反映在AGE法的表征结果中,此外,对于透明质酸在溶液中分子构象的表征,所有表征手段均给出了一致的结果。通过对透明质酸的经验刚性参数B的比较分析可以得到,透明质酸在水溶液中呈半刚性构象,并且低分子量透明质酸比高分子量透明质酸的刚性更加显著,通过对AGE法所得分子量分布的峰态参数的分析可知,低分子量的透明质酸比高分子量的透明质酸的分子间相互作用或缔合能力更强,这与其分子更具刚性的结果相吻合。通过构象图的分析得出透明质酸存在于θ溶剂(0.1 M NaCl)中,成无扰线团构象。通过蠕虫状链圆筒模型得到透明质酸在0.1 M NaCl溶液中的单位围长摩尔质量ML为750 nm-1,持久长度q为20.8 nm,分子链直径d为1.03 nm。以上结果表明,在所研究的分子量范围内,透明质酸在0.1 M NaCl中为蠕虫状链,更加确切为分子链呈半刚性线团构象。至于透明质酸的分子量分布,SEC-MALLS和AFlFFF-MALLS可以对Mw < 1×106的样品进行准确的表征,并且升高分离温度可以提高SEC-MALLS法对Mw > 1×106的样品的分离效果,但是多分散系数的表征结果显示AGE法要优于上述两种表征手段。此外,对于Mw < 1×106的样品来说,SEC-MALLS和AFlFFF-MALLS法表征的分子量分布曲线的形态与AGE法一致。
采用线性Maxwell本构粘弹模型以及非线性Phan-Thien Tanner (PTT)和Giesekus粘弹本构模型对透明质酸溶液的线性和非线性粘弹性行为分别进行了数值模拟。结果表明,三种模型的理论模拟值均能与实验数据很好地吻合,上述模型能够定量描述透明质酸溶液的流变学行为。此外,透明质酸溶液体系的松弛行为具有浓度依赖性,随透明质酸浓度的降低,体系平均松弛时间减小。在所研究的浓度范围内,透明质酸溶液均符合Cox-Merz规则,此时PTT和Giesekus两模型均能定量描述透明质酸溶液的剪切变稀行为。
盐离子可以影响透明质酸溶液的流变学性质。当透明质酸溶液中存在1 M具有较强盐析性质的SO_4~(2-)时,溶液的动态模量和稳态粘度比存在等浓度的Cl-或NO3-时要高,符合Hofmeister序列盐的排列顺序,说明SO_4~(2-)可以使溶液中透明质酸分子间产生相对强的相互作用,并推测透明质酸分子具有一定的疏水性。通过触变环实验可知10 mg/ml透明质酸水溶液及其盐溶液不具有触变性。
发现了透明质酸/玉米纤维胶混合溶液体系中存在协同增粘作用,并申请了专利。此外,利用粘度协同系数对不同配比的透明质酸/玉米纤维胶混合溶液的协同增粘程度进行了定量描述,发现混合溶液的粘度协同系数随着混合溶液中玉米纤维胶浓度的上升而增大,随着剪切速率的增大而减小,即混合溶液的协同增粘效果可以通过改变上述参数进行控制。
发现当透明质酸浓度为10 mg/ml时,透明质酸/阿拉伯胶混合溶液在阿拉伯胶浓度为30 mg/ml时不会产生相分离,在阿拉伯胶浓度达到60 mg/ml时会产生相分离现象。通过宏观观察、流变和光学显微镜实验确定,透明质酸/阿拉伯胶混合溶液产生相分离后,上层液相为透明质酸富集相,下层液相为阿拉伯胶富集相。透明质酸/阿拉伯胶混合溶液产生相分离的机理可能是因为透明质酸和阿拉伯胶同为聚阴离子,以及溶液混合过程中较小所致。此外,无论透明质酸/阿拉伯胶混合溶液是否产生相分离,溶液的稳态剪切粘度都随着混合溶液中阿拉伯胶含量的增加而降低,通过对比透明质酸/聚酰胺-胺混合溶液可知,溶液粘度的降低主要由阿拉伯胶的分子成球形构象及球形胶束状态所致。
流变学、偏光显微镜、光学显微镜、SEM、XRD、DSC和FTIR光谱学的实验结果显示,通过冷冻解冻的方法形成的透明质酸冷冻解冻凝胶属于物理凝胶的范畴,凝胶的三维结构由网络结构接合区中的多重的分子间氢键相互作用所稳定。由于冷冻过程中溶液中的水转变成冰使得透明质酸的浓度升高,因此使透明质酸分子链被迫进行排列,进而形成了并排的分子间缔合,这种缔合可以在解冻过程中继续保持,成为冷冻解冻凝胶网络结构的接合区。透明质酸阴离子的质子化对透明质酸分子链间的缔合来说是必需的,此时透明质酸分子链上-COOH和-NHCOCH3基团间的氢键相互作用在这种缔合的形成过程中扮演了重要的角色。尽管没有发现类似于聚乙烯醇冷冻解冻凝胶中的微结晶区的结构,但是在透明质酸冷冻解冻凝胶中氢键相互作用的特征却得到了实验的证实。基于透明质酸分子链间氢键相互作用可能存在的形式,推断透明质酸冷冻解冻凝胶中的分子链缔合是分散的并且缺少形成微结晶区所需的规整性和完善度,因此无法有效地构建出具有可探测性的微结晶区。此外发现了当在初始透明质酸溶液中加入硫酸钠的浓度达到1 M时,溶液调酸后不进行冷冻解冻处理也可形成透明质酸凝胶。
通过荧光探针法、疏水作用色谱法和疏水吸附法证明了颇具争议的透明质酸疏水性的存在,并且透明质酸分子的疏水相互作用会随着透明质酸溶液浓度的上升而增大。
Mucopolysaccharide is a kind of polysaccharides that widely exists in the animal‘s body. Except the glycogen that is metabolized as the energy, all the polysaccharides in the animal‘s body belong to the mucopolysaccharide. Among them hyaluronan (HA) is the most representative mucopolysaccharide, because the HA was supposed to be the only polysaccharide that exists in almost all animal species, from bacteria to human being. HA has the multiple physiological functions and excellent physicochemical properties. Moreover, HA has been permitted to be used in food by China's Ministry of Health. Nowadays, HA is widely used in medicine, food and cosmetic industry. With the deepening of the research on HA, it also shows a great application potential of HA in the areas of tissue engineering, nanomaterials, etc.
HA is composed of a repeating disaccharide unit of glucuronic acid and N-acetyl-glucosamine. Its molecular weight can be as high as several millions. The physiological functions and application properties of HA are closely related to its molecular parameters, such as the molecular weight, molecular weight distribution; In addition, the viscoelasticity is also one of the important characteristics of HA. The physiological functions of HA, such as maintaining the shape of organ, lubricating joint and absorbing shock, are closely related to its viscoelastic properties; Gelation property is one of the main characteristics of some polysaccharides. For preparation of HA gel, how to maintain the natural biocompatibility and improve the application safety is the focus of the related research. In this thesis, the molecular parameters of HA were detailedly characterized by using multiple-detection techniques, and the rheological properties and physical cryogel of HA were systematically studied. The main content and the conclusions are as follows:
A comparative study using different characterization techniques was carried out to measure the molecular weight, molecular weight distribution and conformational properties of HA. The results show that Capillary viscometry, CG-MALLS, SEC-MALLS, AFlFFF-MALLS and horizontal AGE all provided accurate measurements when Mw of HA was below approximately 2×10~6. A small and negligible discrepancy could be accounted for by the effect of filtration applied in respective methods. When Mw > 2×10~6, capillary viscometry, CG-MALLS, SEC-MALLS and AFlFFF-MALLS gave underestimated values of Mw in comparison with AGE technique. This was attributed to the presence of significant amount of large molecular association or aggregates that were not properly measured in the capillary viscometric and light scattering techniques but were reflected in AGE. Moreover, the techniques all provided consistent results on the conformational properties of HA in solution. The comparative analysis of the empirical rigid parameters B shows that HA adopts a semi-rigid conformation in the solution, and the low molecular weight HA shows more rigidity than high molecular weight HA. The analysis of two statistic parameters, skewness and kurtosis, of the molecular distribution of HA from AGE implies that the association ability of low molecular weight HA is stronger than high molecular weight HA, which is in agreement with the result that the low molecular weight HA is more rigid. The analysis of the conformation plot indicates an unperturbed coil conformation of HA inθsolvent (0.1 M NaCl). On the basis of unperturbed wormlike chain models, the molecular parameters of HA were estimated as follows: the molar mass per unit contour length ML is 750 nm-1, persistence length q is 20.8 nm, and molecular diameter d is 1.03 nm. The above results point out a worm-like chain of HA, more appropriately a semi-rigid coil conformation in the studied molecular weight ranges. With respect to the molecular weight distribution, AGE seemed to be superior to other techniques in measuring polydispersity, although SEC-MALLS and AFlFFF-MALLS were capable of measuring the samples with Mw < 1×10~6 and SEC-MALLS showed improved separation at elevated temperatures for Mw >1×10~6. Furthermore, the shape of distribution profiles measured by SEC-MALLS and AFlFFF-MALLS was nearly identical to that measured by AGE when Mw < 1×10~6.
Three constitutive models, including the linear Maxwell viscoelastic model, non-linear Phan-Thien Tanner (PTT) model and Giseskus model, were employed to describe the linear/non-linear viscoelastic behavior of HA solutions at different concentrations. The calculated results show that all simulated curves calculated from three constitutive model could fit the experiment data quite well, indicating the abovementioned models are competent to describe quantitatively the rheological behavior of HA solution. In addition, the relaxation behavior of HA solution is dependent on the concentration. The average relaxation time shortens with the decreasing concentration of HA solution. It was further found that the HA solutions obeyed the Cox-Merz rule at studied concentration range. Both PTT and Giseskus models are capable of predicting the shear thinning behavior of the concentrated HA solutions.
Salt ions can affect the rheological properties of HA solution. The dynamic moduli and steady-state viscosity of HA solution in presence of strong salting-out salt (1 M SO42-) is higher than those in presence of same concentration of Cl- or NO3-, which obeys the order of Hofmeister salt sequence. This suggests that SO42- can promote the molecular interaction of HA in solution. The existence of certain hydrophobicity of HA was also presumed. The thixotropic rings experiment shows that 10 mg/ml HA aqueous/salt solution have no thixotropy.
The interesting viscous synergism was observed in HA/CFG (corn fiber gum) mixture. Based on this discovery, a patent was applied. Moreover, the degree of viscous synergism in different HA/CFG solutions was quantified by using viscosity synergism index. The result shows that the viscous synergism index increases with the increasing concentration of CFG, and decreases with the increasing shear rate. In other words, the synergistic interaction between the mixed HA and CFG components could be effectively modulated by changing the shear rate and the composition of the mixture.
When the concentration of HA was fixed at 10 mg/ml, the HA/GA (gum arabic) mixture is not phase separated at the GA concentration of 30 mg/ml. However, the phase separation was observed when GA concentration reaches 60 mg/ml. The macroscopic observation, rheological and optical microscopic measurements show that, after the phase separation of HA/GA mixture, the upper phase is the HA enriched phase and the lower phase is GA enriched phase. The phase separation of HA/GA mixture is probably due to the polyanion nature of both HA and GA, and the small in mixing process. In addition, no matter the phase separation occurred or not, the steady-state shear viscosity of HA/GA mixture is reduced with the increasing of GA concentration. Taking into account the rheological properties of HA/PAMAM mixture, the reduction of the viscosity of the HA/GA mixture is mainly caused by the spherical conformation and the formation of spherical micelle of GA molecules.
Based on the experimental results from dynamic rheometry, polarizing and optical microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and FTIR spectroscopy, the obtained HA cryogels after the freeze-thawing cycling undoubtedly belong to the category of physically crosslinked gels whose three dimensional structure is stabilized mainly by multiple interchain hydrogen bonds in the junction zones of the polymeric network. Forced alignment of HA chains as the polymer concentration is increased by conversion of water to ice may provide a mechanism for the formation of side-by-side associations, which then remain intact on thawing, as the junction zones of the cryogel.
Protonation of the polyanion of HA is indispensable to the association of HA chains where hydrogen bonding between groups of -COOH and -NHCOCH3 in HA chains play a crucial role on such bridging. Although cryostructurates in HA cryogels (zones of microcrystallinity) resembling the much more well-studied cryogels on the basis of synthetic polymer, poly(vinyl alcohol) were not found, the feature of hydrogen bonding in HA cryogel were experimentally evident. Based on the different patterns of possible hydrogen bonding between HA chains, it may be reasonable to assume that the intermolecular links in cryogles are discrete, lacking regularity and perfection to form microcrystalline zones, detectable microcrystalline zones thus unable to be constructed effectively.
A controversial property, hydrophobicity, of HA was evidenced by fluorescence probe, hydrophobic interaction chromatography and hydrophobic adsorption experiments. In addition, it is found that the hydrophobic interaction increases with the increasing HA concentration.
透明质酸由葡萄糖醛酸和乙酰氨基葡萄糖双糖重复单元组成,其分子量可高达上百万,透明质酸的生理功能和应用特性与其分子量、分子量分布等分子参数密切相关;此外,粘弹性也是透明质酸重要的特性之一,透明质酸在体内所发挥的维持组织形态、润滑关节和缓冲应力等作用均与其粘弹性密切相关;凝胶化作用是一些多糖大分子最主要的特性之一,在制备透明质酸凝胶时如何保持其天然的生物相容性同时提高其应用的安全性是相关研究的焦点问题。本论文利用多种技术手段对透明质酸的分子参数进行了详细的表征,并对透明质酸的流变学性质及其冷冻解冻物理凝胶进行了系统的研究,主要内容及结论如下:
对一系列透明质酸样品的分子参数(分子量、分子量分布、分子尺寸及分子构象等)的多手段表征进行了系统性的对比研究。结果显示,对于Mw低于约2×106的样品来说,粘度法、CG-MALLS、SEC-MALLS、AFlFFF-MALLS和AGE法均能给出准确的表征结果,表征结果所存在的略微差异是由于过滤过程应用于不同的表征手段。对于Mw高于约2×106的样品来说,粘度法、CG-MALLS、SEC-MALLS、AFlFFF-MALLS给出了低于AGE法的Mw表征结果,这是由于透明质酸溶液中存在大尺寸的分子缔合或聚集所引起的,而这种分子聚集体无法用粘度法和静态光散射法进行准确的表征,但是可以反映在AGE法的表征结果中,此外,对于透明质酸在溶液中分子构象的表征,所有表征手段均给出了一致的结果。通过对透明质酸的经验刚性参数B的比较分析可以得到,透明质酸在水溶液中呈半刚性构象,并且低分子量透明质酸比高分子量透明质酸的刚性更加显著,通过对AGE法所得分子量分布的峰态参数的分析可知,低分子量的透明质酸比高分子量的透明质酸的分子间相互作用或缔合能力更强,这与其分子更具刚性的结果相吻合。通过构象图的分析得出透明质酸存在于θ溶剂(0.1 M NaCl)中,成无扰线团构象。通过蠕虫状链圆筒模型得到透明质酸在0.1 M NaCl溶液中的单位围长摩尔质量ML为750 nm-1,持久长度q为20.8 nm,分子链直径d为1.03 nm。以上结果表明,在所研究的分子量范围内,透明质酸在0.1 M NaCl中为蠕虫状链,更加确切为分子链呈半刚性线团构象。至于透明质酸的分子量分布,SEC-MALLS和AFlFFF-MALLS可以对Mw < 1×106的样品进行准确的表征,并且升高分离温度可以提高SEC-MALLS法对Mw > 1×106的样品的分离效果,但是多分散系数的表征结果显示AGE法要优于上述两种表征手段。此外,对于Mw < 1×106的样品来说,SEC-MALLS和AFlFFF-MALLS法表征的分子量分布曲线的形态与AGE法一致。
采用线性Maxwell本构粘弹模型以及非线性Phan-Thien Tanner (PTT)和Giesekus粘弹本构模型对透明质酸溶液的线性和非线性粘弹性行为分别进行了数值模拟。结果表明,三种模型的理论模拟值均能与实验数据很好地吻合,上述模型能够定量描述透明质酸溶液的流变学行为。此外,透明质酸溶液体系的松弛行为具有浓度依赖性,随透明质酸浓度的降低,体系平均松弛时间减小。在所研究的浓度范围内,透明质酸溶液均符合Cox-Merz规则,此时PTT和Giesekus两模型均能定量描述透明质酸溶液的剪切变稀行为。
盐离子可以影响透明质酸溶液的流变学性质。当透明质酸溶液中存在1 M具有较强盐析性质的SO_4~(2-)时,溶液的动态模量和稳态粘度比存在等浓度的Cl-或NO3-时要高,符合Hofmeister序列盐的排列顺序,说明SO_4~(2-)可以使溶液中透明质酸分子间产生相对强的相互作用,并推测透明质酸分子具有一定的疏水性。通过触变环实验可知10 mg/ml透明质酸水溶液及其盐溶液不具有触变性。
发现了透明质酸/玉米纤维胶混合溶液体系中存在协同增粘作用,并申请了专利。此外,利用粘度协同系数对不同配比的透明质酸/玉米纤维胶混合溶液的协同增粘程度进行了定量描述,发现混合溶液的粘度协同系数随着混合溶液中玉米纤维胶浓度的上升而增大,随着剪切速率的增大而减小,即混合溶液的协同增粘效果可以通过改变上述参数进行控制。
发现当透明质酸浓度为10 mg/ml时,透明质酸/阿拉伯胶混合溶液在阿拉伯胶浓度为30 mg/ml时不会产生相分离,在阿拉伯胶浓度达到60 mg/ml时会产生相分离现象。通过宏观观察、流变和光学显微镜实验确定,透明质酸/阿拉伯胶混合溶液产生相分离后,上层液相为透明质酸富集相,下层液相为阿拉伯胶富集相。透明质酸/阿拉伯胶混合溶液产生相分离的机理可能是因为透明质酸和阿拉伯胶同为聚阴离子,以及溶液混合过程中较小所致。此外,无论透明质酸/阿拉伯胶混合溶液是否产生相分离,溶液的稳态剪切粘度都随着混合溶液中阿拉伯胶含量的增加而降低,通过对比透明质酸/聚酰胺-胺混合溶液可知,溶液粘度的降低主要由阿拉伯胶的分子成球形构象及球形胶束状态所致。
流变学、偏光显微镜、光学显微镜、SEM、XRD、DSC和FTIR光谱学的实验结果显示,通过冷冻解冻的方法形成的透明质酸冷冻解冻凝胶属于物理凝胶的范畴,凝胶的三维结构由网络结构接合区中的多重的分子间氢键相互作用所稳定。由于冷冻过程中溶液中的水转变成冰使得透明质酸的浓度升高,因此使透明质酸分子链被迫进行排列,进而形成了并排的分子间缔合,这种缔合可以在解冻过程中继续保持,成为冷冻解冻凝胶网络结构的接合区。透明质酸阴离子的质子化对透明质酸分子链间的缔合来说是必需的,此时透明质酸分子链上-COOH和-NHCOCH3基团间的氢键相互作用在这种缔合的形成过程中扮演了重要的角色。尽管没有发现类似于聚乙烯醇冷冻解冻凝胶中的微结晶区的结构,但是在透明质酸冷冻解冻凝胶中氢键相互作用的特征却得到了实验的证实。基于透明质酸分子链间氢键相互作用可能存在的形式,推断透明质酸冷冻解冻凝胶中的分子链缔合是分散的并且缺少形成微结晶区所需的规整性和完善度,因此无法有效地构建出具有可探测性的微结晶区。此外发现了当在初始透明质酸溶液中加入硫酸钠的浓度达到1 M时,溶液调酸后不进行冷冻解冻处理也可形成透明质酸凝胶。
通过荧光探针法、疏水作用色谱法和疏水吸附法证明了颇具争议的透明质酸疏水性的存在,并且透明质酸分子的疏水相互作用会随着透明质酸溶液浓度的上升而增大。
Mucopolysaccharide is a kind of polysaccharides that widely exists in the animal‘s body. Except the glycogen that is metabolized as the energy, all the polysaccharides in the animal‘s body belong to the mucopolysaccharide. Among them hyaluronan (HA) is the most representative mucopolysaccharide, because the HA was supposed to be the only polysaccharide that exists in almost all animal species, from bacteria to human being. HA has the multiple physiological functions and excellent physicochemical properties. Moreover, HA has been permitted to be used in food by China's Ministry of Health. Nowadays, HA is widely used in medicine, food and cosmetic industry. With the deepening of the research on HA, it also shows a great application potential of HA in the areas of tissue engineering, nanomaterials, etc.
HA is composed of a repeating disaccharide unit of glucuronic acid and N-acetyl-glucosamine. Its molecular weight can be as high as several millions. The physiological functions and application properties of HA are closely related to its molecular parameters, such as the molecular weight, molecular weight distribution; In addition, the viscoelasticity is also one of the important characteristics of HA. The physiological functions of HA, such as maintaining the shape of organ, lubricating joint and absorbing shock, are closely related to its viscoelastic properties; Gelation property is one of the main characteristics of some polysaccharides. For preparation of HA gel, how to maintain the natural biocompatibility and improve the application safety is the focus of the related research. In this thesis, the molecular parameters of HA were detailedly characterized by using multiple-detection techniques, and the rheological properties and physical cryogel of HA were systematically studied. The main content and the conclusions are as follows:
A comparative study using different characterization techniques was carried out to measure the molecular weight, molecular weight distribution and conformational properties of HA. The results show that Capillary viscometry, CG-MALLS, SEC-MALLS, AFlFFF-MALLS and horizontal AGE all provided accurate measurements when Mw of HA was below approximately 2×10~6. A small and negligible discrepancy could be accounted for by the effect of filtration applied in respective methods. When Mw > 2×10~6, capillary viscometry, CG-MALLS, SEC-MALLS and AFlFFF-MALLS gave underestimated values of Mw in comparison with AGE technique. This was attributed to the presence of significant amount of large molecular association or aggregates that were not properly measured in the capillary viscometric and light scattering techniques but were reflected in AGE. Moreover, the techniques all provided consistent results on the conformational properties of HA in solution. The comparative analysis of the empirical rigid parameters B shows that HA adopts a semi-rigid conformation in the solution, and the low molecular weight HA shows more rigidity than high molecular weight HA. The analysis of two statistic parameters, skewness and kurtosis, of the molecular distribution of HA from AGE implies that the association ability of low molecular weight HA is stronger than high molecular weight HA, which is in agreement with the result that the low molecular weight HA is more rigid. The analysis of the conformation plot indicates an unperturbed coil conformation of HA inθsolvent (0.1 M NaCl). On the basis of unperturbed wormlike chain models, the molecular parameters of HA were estimated as follows: the molar mass per unit contour length ML is 750 nm-1, persistence length q is 20.8 nm, and molecular diameter d is 1.03 nm. The above results point out a worm-like chain of HA, more appropriately a semi-rigid coil conformation in the studied molecular weight ranges. With respect to the molecular weight distribution, AGE seemed to be superior to other techniques in measuring polydispersity, although SEC-MALLS and AFlFFF-MALLS were capable of measuring the samples with Mw < 1×10~6 and SEC-MALLS showed improved separation at elevated temperatures for Mw >1×10~6. Furthermore, the shape of distribution profiles measured by SEC-MALLS and AFlFFF-MALLS was nearly identical to that measured by AGE when Mw < 1×10~6.
Three constitutive models, including the linear Maxwell viscoelastic model, non-linear Phan-Thien Tanner (PTT) model and Giseskus model, were employed to describe the linear/non-linear viscoelastic behavior of HA solutions at different concentrations. The calculated results show that all simulated curves calculated from three constitutive model could fit the experiment data quite well, indicating the abovementioned models are competent to describe quantitatively the rheological behavior of HA solution. In addition, the relaxation behavior of HA solution is dependent on the concentration. The average relaxation time shortens with the decreasing concentration of HA solution. It was further found that the HA solutions obeyed the Cox-Merz rule at studied concentration range. Both PTT and Giseskus models are capable of predicting the shear thinning behavior of the concentrated HA solutions.
Salt ions can affect the rheological properties of HA solution. The dynamic moduli and steady-state viscosity of HA solution in presence of strong salting-out salt (1 M SO42-) is higher than those in presence of same concentration of Cl- or NO3-, which obeys the order of Hofmeister salt sequence. This suggests that SO42- can promote the molecular interaction of HA in solution. The existence of certain hydrophobicity of HA was also presumed. The thixotropic rings experiment shows that 10 mg/ml HA aqueous/salt solution have no thixotropy.
The interesting viscous synergism was observed in HA/CFG (corn fiber gum) mixture. Based on this discovery, a patent was applied. Moreover, the degree of viscous synergism in different HA/CFG solutions was quantified by using viscosity synergism index. The result shows that the viscous synergism index increases with the increasing concentration of CFG, and decreases with the increasing shear rate. In other words, the synergistic interaction between the mixed HA and CFG components could be effectively modulated by changing the shear rate and the composition of the mixture.
When the concentration of HA was fixed at 10 mg/ml, the HA/GA (gum arabic) mixture is not phase separated at the GA concentration of 30 mg/ml. However, the phase separation was observed when GA concentration reaches 60 mg/ml. The macroscopic observation, rheological and optical microscopic measurements show that, after the phase separation of HA/GA mixture, the upper phase is the HA enriched phase and the lower phase is GA enriched phase. The phase separation of HA/GA mixture is probably due to the polyanion nature of both HA and GA, and the small in mixing process. In addition, no matter the phase separation occurred or not, the steady-state shear viscosity of HA/GA mixture is reduced with the increasing of GA concentration. Taking into account the rheological properties of HA/PAMAM mixture, the reduction of the viscosity of the HA/GA mixture is mainly caused by the spherical conformation and the formation of spherical micelle of GA molecules.
Based on the experimental results from dynamic rheometry, polarizing and optical microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and FTIR spectroscopy, the obtained HA cryogels after the freeze-thawing cycling undoubtedly belong to the category of physically crosslinked gels whose three dimensional structure is stabilized mainly by multiple interchain hydrogen bonds in the junction zones of the polymeric network. Forced alignment of HA chains as the polymer concentration is increased by conversion of water to ice may provide a mechanism for the formation of side-by-side associations, which then remain intact on thawing, as the junction zones of the cryogel.
Protonation of the polyanion of HA is indispensable to the association of HA chains where hydrogen bonding between groups of -COOH and -NHCOCH3 in HA chains play a crucial role on such bridging. Although cryostructurates in HA cryogels (zones of microcrystallinity) resembling the much more well-studied cryogels on the basis of synthetic polymer, poly(vinyl alcohol) were not found, the feature of hydrogen bonding in HA cryogel were experimentally evident. Based on the different patterns of possible hydrogen bonding between HA chains, it may be reasonable to assume that the intermolecular links in cryogles are discrete, lacking regularity and perfection to form microcrystalline zones, detectable microcrystalline zones thus unable to be constructed effectively.
A controversial property, hydrophobicity, of HA was evidenced by fluorescence probe, hydrophobic interaction chromatography and hydrophobic adsorption experiments. In addition, it is found that the hydrophobic interaction increases with the increasing HA concentration.
引文
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