Helical Chain Conformation Of A Polysaccharide Isolated From Black Fungus And Its Biomedical Applications

Fungal polysaccharides have a wide range of sources,such as convolution,letinous edodes and black fungus,which are rich in biologic polysaccharides,and show the biological activity of anti-tumor,antiviral,antioxidation and immunoregulation.As we all know,structure determines function.The structure of natural polysaccharides and chain conformation will inevitably affect their biological activities.However,the wide range of polysaccharides and the variety of bonding types make it very difficult for structural analysis.Moreover,the chain conformation of polysaccharides is also very complex.In solution,it has many kinds of chain conformation,such as spherical chain,rigid chain,compliant chain,spiral chain and so on.Therefore,the structure and chain conformation of polysaccharides were determined to provide important information for constructing the structure-activity relationship of polysaccharides.Biologic macromolecules have specific properties of self-assembly,such as the folding and folding of proteins,or the double helix structure of DNA.However,there are few studies on the self-assembly behavior of fungal polysaccharides.Several kinds of fungal polysaccharides,such as scleroglucan,schizophyllum polysaccharide and letinous edodes polysaccharide,are used to load and release guest molecules by the destruction and reconstruction of the triple helix structure.Whether there existed ordered assembly of helical polysaccharides is unknown.Moreover,the biomedical materials based on the self-assembly properties of fungal polysaccharides are rarely reported.Therefore,the aim of this work is to further study the structure,conformation and aggregation structure of fungal polysaccharides through the innovation of characterization methods.At the same time,we further reveal the self-assembly behavior of fungal polysaccharides,and develop biomedical materials based on polysaccharides and explore their potential applications.The main innovations of this thesis include the following points:(1)By light scattering(LLS/DLS),viscosity and atomic force microscopy(AFM),together with molecular dynamics simulation,the triple helix conformation of BFP,a polysaccharide extracted from the fruiting bodies of black fungus,was determined first time.(2)The self-assembly behaviors of BFP in water was explored by using the aggregation induced emission(AIE)fluorescent probe,and the formation mechanism of the dendritic nanotubes(BFP-DNTs)was discussed and the application of the fluorescent dye loaded nanotubes as probe in the biological imaging field was explored.(3)Based on the hydrophobic cavity of BFP nanotubes,a kind of cancer drugs(DOX)was successfully loaded,and the usage for drug delivery and release was also discussed.(4)Successfullyin situ immobilization of silver nanoparticles on the surface of BFP nanotubes,and it is proved that the nanocomposites have good antibacterial properties.(5)BFP is used as reducer,stabilizer and dispersants to synthesize gold nanoparticles.The nanoscale effect of gold nanoparticles is also studied,and the excellent catalytic performance is proved.The main contents and conclusions of this paper are as follows.First of all,the β-1,3-D-glucan was extracted from black fungus by hot salt water.The chemical structureof BFP was determined by ion exchange chromatography(HPAEC),gaschromatography-mass spectrometry(GC-MS)and nuclear magnetic(NMR),and everythree β-1,3-D-glucose residues in the main chain had two β-1,6-D-glucose residues.The conformation parameters of the BFP chain are calculated by light scattering andviscosity combined with solution theory.The Mark-Houwink equation of BFP with Mw ranged from 46×104 g/mol to 216×104 g/mol is[η]= 1.78×10-9 Mw1.6.The calculated molar mass per unit contour length(ML),the persistence length(q),the chain diameter(d)and the pitch distance(h)are about 2448～3000 nm-1、192～260 nm、2.2 nm and 0.37 nm,respectively,which are very close to the conformation parameters of triple helical chain.It is worth noting that the molecular size of BFP is also directly observed by AFM,and the relationship between molecular weight and chain length is established by statistical chain length:Mw(g/mol)= 2212 LAFM,nm+ 79599.Thus,new method for characterization of molecular chains for the rigid polysaccharides could be established directly by AFM measurement.In addition,the whole atomic molecular dynamics(MD)simulation combined with simulated annealing(SA)algorithm is used to predict the minimum energy structure of BFP.The results show that the two twisting angles of the glycoside bonds on the main chain,Φ(H1-C1-03-C3)and Ψ(C1-03-C3-H3),are 45.51 and-16.97,and the BFP molecular chain is the most stable and can form a very stable triple-helix structure,and the energy of each chain is lower than that of the single strand.This means that in nature,triple-helix structures are more likely to exist than single chains for black fungus polysaccharides.At the same time,the structure of triple-helix structure is more compact than that of single helix,and many hydrogen bonds are formed between different spiral chains,which makes the helical structure very stable.Therefore,in this chapter,the structure and conformation of the glucan of black fungus is characterized by a variety of methods,which provides a valuable theoretical basis for the multilevel structure characterization of natural polysaccharide.Based on the rigid chain conformation of BFP,we studied the self-assembly behaviors of BFP.It was found that the polysaccharides showed monodisperse-chain distribution under extremely dilute conditions.Once the concentration of the solution increased,the number of molecular chains increased and the molecular chains gradually tended to form a lamella structure.Continuing to increase the concentration,it would induce the accumulation of molecular chains and even gather into dendritic nanotubes(BFP-DNTs).By transmission electron microscopy(TEM)and scanning electron microscopy(SEM),it was proved that the formation of the fiber was formed by the curling of the polysaccharide molecular chains.At the same time,the introduction of aggregation induced emission(AIE)fluorescent probe proves that there is a hydrophobic cavity in the nanotubes,that is,the addition of BFP-DNTs induces the aggregation and luminescence of dye molecules.It is worth noting that the BFP nanotube can encapsulate the guest molecules,and the dendritic structure leads to the increase of the concentration of the contained objects,thus achieving the effect of highly concentrated target molecules.Moreover,BFP-DNTs that loaded with hydrophobic dye molecules has lower biological toxicity and stronger fluorescence intensity than pure dye molecules.Moreover,the biological imaging test in nude mice showed that the polysaccharide maintained a longer fluorescence duration in vivo(18 days)after inclusion of dye molecules.Therefore,this work provides a new idea for the carrier system in the field of biological imaging.To detect the loading behaviors of BFP nanotubes which possessed hydrophobic cavity,doxorubicin(DOX),a typical anticancer drug is used to detect the loading behaviors of BFP nanotubes.The experimental results show that the BFP-DNTs can reach a high loading rate(34%)and entrapment efficiency(68%)of DOX,which is closely related to the tree structure and hollow cavity of the nanotubes.At the same time,the study showed that the nanotube could protect DOX from releasing in normal tissues(pH=7.4),and could be released only at the lesion site(pH=5.0),thus achieving therapeutic effect without damaging the body’s healthy tissue.This indicates that BFP has good biocompatibility and anti-tumor activity.This new type of nontoxic side effect polysaccharide dendritic nanotube(BFP-DNTs)can provide a new effective way for drug delivery system.Based on the hydrophobic cavity,tubular structure and abundant hydroxyl groups on the surface of BFP nanotubes,the scaffolds for nanoparticles synthesized were established.A simple and mild chemical reduction method was used to produce silver nanoparticles with smaller size(14～23 nm)with uniform size distribution on the substrate of BFP nanotubes.The loading capacity of silver nanoparticles is controllable,ranging from 6%to 51%.The silver nanoparticles were uniformly dispersed on the surface of the nanotubes,confirmed by EDS,TEM and SEM results.In addition,we evaluated the antibacterial properties of silver nanoparticles/nanotubes complexes(BFP-Ag),and the antibacterial properties increased with the increase of silver content.Therefore,the BFP-Ag with good antibacterial activity and good stability will be applied in the field of antibacterial.Moreover,BFP was used as reducing agents,dispersants and stabilizers to obtain gold nanoparticles with smaller size and uniform dispersion.It is proved that the shape(band or sphere)and size of gold nanoparticles(10 nm～19 nm)can be effectively controlled by changing the reaction temperature,reaction time and the concentration of BFP and Au3+.Gold nanoparticles/nanotubes complexes(BFP-Au)can effectively catalyze the reduction of p-nitrophenol(4-NP)and confirm the nanoscale effect of gold nanoparticles.This chapter provides new ideas for the synthesis and dispersion of nanoparticles,and expands the potential applications of polysaccharides in the field of nanomaterials.The above results were the first determination of the chemical structure,molecular weight,molecular size and the single helical rigid chain conformation of black fungus in solution.Based on the conformation of the polysaccharide rigid chain,its self-assembly behaviors in water were discussed,and dendritic nanotubes were constructed.Meanwhile,based on the special structure of BFP,four functional biomedical materials were constructed,and their applications were preliminarily evaluated.This paper provides a new method and new idea for the structure and conformation characterization of polysaccharides,and provides important scientific basis for the biomaterials construction.Therefore,it has important academic value and application prospect.