Structure, Chain Conformation And Functional Modification Of Hyperbranched Polysaccharide

Fungal polysaccharides are called as excellent immunomodulators, which can improve the body's immune defenses without any damage to the normal cell. It has been reported that the bioactivities of polysaccharides are correlated closely to their molecular structure and chain conformation in solution. Due to the unique branched topology, hyperbranched polysaccharide exhibits spherical chain conformation in solution. Compared with the linear polysaccharides, hyperbranched polysaccharides show more excellent performances, and have great potential applications in many areas. In this thesis, two kinds of hyperbranched polysaccharides were extracted and purified from the sclerotia of Pleurotus tuber-regium, the chemical structure were ascertained using characterization methods and instruments for chemical analysis. Moreover, their molecular parameters and chain conformation were determined based on the laser light scattering, viscosity and atomic force microscopy, combined with the theory of polymer solution. Studies on the functional application were performed on the basis of their structure features of the hyperbranched polysaccharides. Therefore, this work involves interdisciplinary fields of polymer physics, glycochemistry, nanoscience and materials science, and also one of the international research frontiers of polymer science.The innovative points of this work are as follows. (1) It has been confirmed that water-soluble polysaccharide (HBP) extracted from the sclerotia of Pleurotus tuber-regium was a hyperbranchedβ-D-glucan, the aggregation mechanism of HBP in aqueous solution was clarified for the first time. (2) Uniform and well-dispersed selenium nanoparticles in hyperbranched polysaccharide aqueous solution were successfully constructed by redox for the first time. The structure, stability and formation mechanism of Se-HBP nanocomposites were clarified. (3) Non-planar step-wise adsorption of polysaccharide-based polyelectrolytes were performed onto charged colloidal ZnO microspheres in solution to create multilayer hollow microcapsules; (4) It has been firstly revealed that water-insoluble polysaccharide (PTR) was a hyperbranchedβ-D-glucan, which exhibited spherical chain conformation in solution.The main contents and conclusions in this thesis are divided into the following parts. A water-soluble polysaccharide was isolated from the sclerotia of Pleurotus tuber-regium by 0.9% NaCl at 120℃under high pressure. The chemical structure was analyzed by using infrared spectroscopy (IR), ultraviolet spectroscopy (UV) and nuclear magnetic resonance spectroscopy (13C NMR). Fluorescence spectroscopy (FS) was used to determine the critical aggregation concentration (CAC) of HBP in aqueous solution. Meanwhile, intrinsic viscosity ([η]), weight-average molecular weight (Mw) and hydrodynamic radius (Rh) of HBP in solution with different ratios DMSO to water were measured with the viscometer and dynamic laser light scattering (DLS), to reveal the changes of the molecular size in solution and aggregation behavior. The results indicated that HBP is a hyperbranchedβ-D-glucan withβ-(1→6),β-(1→4),β-(1→3) andβ-(1,4,6)-linked residues, with degree of branching (DB) of 57.6%, and its weight-average molecular weight (Mw) is about 1.11 x 106 g/ mol. HBP is soluble in water, because of the large numbers of branched chains or side groups, leading to the enhancement of solubility in water. The polysaccharide was very easy to aggregate in aqueous solution with the critical aggregation concentration of 0.35 mg/mL and the apparent aggregation number of 13, whereas individual molecules of HBP occurred in DMSO. Due to the hydrophilic hydroxyl end-group and the hydrophobic microzones, HBP exhibited amphiphilic, leading to the existence of the aggregates with tight structure in aqueous solution. However, more loosely single-chain existed in DMSO because of the hydrophobic interaction. Furthermore, TEM results showed that all of the aggregates and individual molecules exhibited spherical shapes in the solutions. The molecular size and intrinsic viscosity of HBP polysaccharides changed in DMSO/water with the water content, reflecting the changes of the intermolecular hydrogen bonding interaction.Usually, natural hyperbranched polysaccharides (HBP) have the large number of terminal hydroxyl groups and high specific surface area, leading to a strong adsorption on some elements. Selenium nanoparticles (SeNPs) with mean sizes of 24 nm were created successfully in water system via a novel synthetic pathway of HBP-capped reaction sites under extremely safe condition. The results from TEM, AFM and light scattering revealed that SeNPs were capped by the HBP molecules to prevent aggregation of the grown nanoparticles, leading a good dispersion of the Se nanoparticles in water. HBP played an important role as capping agent in the formation and stabilization of the Se-HBP nanocomposites, as a result of the strong physical adsorption of HBP on SeNPs surfaces. The hyperbranched polysaccharide supplied not only reaction sites for the creation of Se nanoparticles, but also a shell to protect nanoparticles structure, leading to water-dispersible nanoparticles. This was a simple method for the preparation of the organic-inorganic hybrid composites with high safety, biocompatibility and biodegradability, which is very important in the field of life science and technology.A water-insoluble polysaccharide (GP) was extracted from the fruiting body of Ganoderma lucidum. Carboxymethyl polysaccharides (CMGP) and quaternized polysaccharides (QGP) were prepared by carboxymethylation and quaternization reaction in NaOH aqueous solution. The structure of the polysaccharide and its derivatives were determined by IR, NMR and viscosity. The results indicated that GP was a linearβ-(1→3)-D-glucan, and the two derivatives displayed a typical polyelectrolyte effect in aqueous solution. Zinc oxide (ZnO) microspheres were used as templates, and the polysaccharide-based polyelectrolytes QGP, CMGP were self-assemblied successfully to form the composite microspheres. The structure and size of ZnO microspheres and the composite microspheres was studied by TEM, SEM,ζ-potential measurement and DLS. The results showed that the size of ZnO microsphere was about 712 nm. The core-shell composite microspheres with mean shell thickness composite microspheres of 20～24nm were fabricated by consecutively assembling CMGP and QGP onto the ZnO colloidal particles. The integrity-structural and ellipsoidal hollow microcapsules were obtained by removing the templated colloid by chemical methods. This work provided the possible application of natural polysaccharide in the fields of drug delivery and controlled release.A water-insoluble sample (PTR), extracted from the sclerotia of Pleurotus tuber-rigium by using 2 M NaOH/0.01 M NaBH4 aqueous solution, was analyzed by IR, one-and two-dimensional 1H and 13C NMR. The results indicated that PTR was hyperbranchedβ-(1→3)-D-glucan with residuals branched at C3, C2, C4, and C6 positions. The sample PTR was fractionated by non-solvent addition method into eight fractions and their [η] and Mw were determined by LLS and viscometry. In accordance with the polymer solution theory, the dependence of [77] on Mw was established as the Mark-Houwink equation:[η]= 0.04w0.45±0.02 in the Mwrange from 1.29 x 106 to 5.01 x 107 for PTR in DMSO at 25℃. The results indicated that the hyperbranched polysaccharide existed as a compact chain conformation with sphere-like structure in DMSO. The unique molecular structure made the hyperbranched polysaccharide a strong overlap tendency to form three-dimensional network. Based on the structural features, PTR was reacted with chitin in NaOH/urea aqueous solution to obtaine chemical cross-linking chitin-PTR composite hydrogels. The formation and structure of the hydrogels were determined by IR, X-ray diffraction (XRD), solid NMR (CP/MAS), thermal gravimetric analysis (TG). The performances of the composite hydrogels were studied by SEM, equilibrium swelling and cell culture test. The results showed that in the composite hydrogel, chitin contributed to support the pore wall whereas PTR acted as an expander of the pore size. The composite hydrogel showed well-defined three-dimensional porous network structures and good biocompatibility, which was conducive to cell growth. The composite hydrogel have potential applications in the field of tissue scaffolds.The basic research mentioned above focused on the chemical structure, molecular size and chain conformation in solution of the hyperbranched polysaccharides. At the same time, We revealed the relationship between the structural factors, the performance and functionality of the polysaccharides. These results not only had scientific significance, but also provided a series of valuable data for the exploration and application of the traditional Chinese medicine resources.