Study On The Molecular Characterization, Rheological Properties And Physical Cryogel Of Mucopolysaccharide Hyaluronan

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.