Cationic Polysaccharide For Non-viral Gene Delivery And Its Structure-activity Relationship

Natural polysaccharides,such as chitin and inulin,heparin,are a kind of natural macromolecule material.Natural polysaccharide plays an important role in organism by serving as one of the major structural components of the organism structure and participating in multiple signaling transductions as well.There are many reports about the biological function of natural polysaccharides,such as immune regulation,anti-tumor,anti-aging and antiviral pharmacological.However,the research of natural polysaccharide as gene carrier has just started,and had very little knowledge in the structure-activity relationship.In this study,natural polysaccharides were isolated from mushroom-Pleurotus eryngii and Flammulina velutipes via a series of separation and purification steps.Then,the primary and advanced structure of polysaccharides was analyzed.Amino compounds,ethylenediamine and spermine,were employed to modifiy the natural polysaccharide skeleton surface to obtain positive surface charges for gene condensation and encapsulation.The structure-activity relationship of the natural polysaccharides was also discussed preliminarily based on the transfection efficiency which varied according to the different structures of the cationic polysaccharides.Chapter 1 Application and research progress of natural polysaccharides in gene deliveryThe development of safe and effective gene delivery system is the major challenge of gene treatment and DNA vaccine.Natural polysaccharides have many advantages for gene delivery such as good transfection efficiency,good biological intermiscibility,biodegradability and bioactivity.This study mainly summarized the classification of natural polysaccharides and thier modification,and reviewed the research progress of nature polysaccharides for gene deivery,and predicted their application and development trend in the future.Chapter 2 Extracti on,separati on and purificati on of nat ural pol ysacchari desIn this section,crude polysaccharides were extracted from Pleurotus eryngii or Flammulina velutipes by hot water and then purified by protein removal with chloroacetic acid.One polysaccharide from Pleurotus eryngii(named as PEP)and two polysaccharides from Flammulina velutipes(names as FVP-D and FVP-X)were further purified by macroporous resin D101,DEAE-52 resin fiber,Sephadex G100 column in sequence.Molecular weight,monosaccharide composition,linkages between monosaccharide units and configuration of monosaccharides were performed to characterize the refined polysaccharides.Congo red and atomic force microscope(AFM)were used to analyze advanced structurs of the polysaccharides.The results showed that the average molecular weight of PEP was 1561.907 KDa,and the major monosaccharide components were glucose,galactose and mannose with the molar ratio of 8.76:1.97:1.The three monosaccharide components were found to possess alpha configuration,the linkages between these monosaccharide units were 1?2 or l?4 connections.Congo red and AFM experiments showed that PEP,without the helical structure,was ? glucan heteropolysaccharide with a spherical shape.The average molecular weight of FVP-D was 198.198 KDa,and the major monosaccharide components were galactose,glucose and arabinose with the molar ratio of 51.36:2.37:15.55.The linkage between the monosaccharide units was 1?3 connections.Congo red and AFM experiments showed that PEP was an arabinose ? galactan with a helical structure and rod-like or spherical shape.Chapter 3 The cationic modification of natural polysaccharides and their characterizationIn this study,natural polysaccharides were chemically grafted with ethylenediamine or spermine using N,N'-Carbonyldiimidazole(CDI)as the crosslinker to obtain six cationicd polysaccharides.The cationic polysaccharides were characterized by FT-IR,1H-NMR,elemental analysis,and size and Zeta potential determination.Monosaccharide composition,linkages between monosaccharide units of the cationicd polysaccharides were also studied.The results showed that the positively charged cationic polysaccharides were successfully prepared(named as PEP-Y,PEP-J,FVP-D-Y,FVP-D-J,FVP-X-Y and FVP-X-J,respectively).PEP-J had the highest Zeta potential(45.8 ± 0.97 Mv)and FVP-X-Y showed the lowest positive charge(21.6 ± 0.2 Mv).The results of monosaccharide composition showed that the cationic polysaccharides retained some of the non-modified monosaccharide components.Chapter 4 The prepartion of cationized polysaccharides for non-viral gene deliveryIn this chapter,cationized polysaccharide/p SOX2 complexes were obtained via electrostatic interaction between the cationized polysaccharideds and the plasmid SOX2.Agarose gel electrophoresis was carried out to examine the combination between the cationized polysaccharides and p SOX2.Dynamic light scattering and Zeta potential instrument was employed to measure the particle size and Zeta potential of the resulting cationized polysaccharide/p SOX2 complexs.AFM was used to observe the morphology of the nanoparticles.The results showed that cationized polysaccharide/p DNA complexes can completely retain plasmid SOX2 and the lowest ratio has no correlation with the Zeta potential of the cationized polysaccharides.It may be related to the spatial structure of cationized polysaccharide.PEP-Y/p SOX2,PEP-J/p SOX2,FVP-D-Y/p SOX and FVP-D-J/p SOX2 complexes were spherical nanoparticles.However,the shape of FVP-X-Y/p SOX and FVP-X-J/p SOX2 complexes were irregular.It was probably because its single helix chain structure and small molecular weight could not form spherical nanoparticles after combining with p SOX2.Chapter 5 The study of transfection efficiency and uptake mechanism of cationic polysaccharide/p SOX2 complexesThe cytotoxicity of the cationic polysaccharide/p SOX2 complexes were investigated by MTT method.Enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative PCR(q RT-PCR)were carried out to measure transfection efficiency of nanoparticles in rat embryonic fibroblasts(REF).The cell uptake mechanism of cationic polysaccharide/p YOYO-1-SOX2 complex was studied by fluorescence microscopy.The results showed that the toxicity of cationic polysaccharide/p SOX2 complexes with different mass ratio was significantly lower than that of positive transfection reagent Lipofectaime 2000 and PEI 25 KDa,and the toxicity of PEP-J/p SOX2 complex was relatively larger.Transfection efficiency results of six cationic polysaccharide/p SOX2 compound were as follows: PEP-J/p SOX2(40:1),FVP-D-J/p SOX2(40:1),PEP-Y/p SOX2(20:1),FVP-D-Y/SOX2(20:1),FVP-X-J/p SOX2(2:1),FVP-X-Y/SOX2(20:1).The transfection efficiency of the cationic polysaccharides were higher than Lipofectaime 2000 and PEI 25 KDa.Preliminary analysis of structure-activity relationship was as follows: transfection efficiency of the spherical shape with high molecular weight,alpha glucan heteroside was higher than that of the middle molecular weight,shape or spherical beta Arabia galactosan,and the single chain type with low molecular weight,alpha glucan heteroside possessed the lowest transfection efficiency.The cellular uptake mechanism study of PEP-J/p SOX2(40:1)and FVP-D-J/p SOX2(2:1)with high transfection efficiency showed that PEP-J/p SOX2(40:1)was uptaked by REF mainly through clathrin mediated endocytosis and caveolin mediated endocytosis,followed by entering endosome lysosome pathway and proceeding intracellular transport under participation of fibrous protein and dynein protein,and intracellular transshipment of FVP-D-J/p SOX2(2:1)complexes was under the participation of cytoskeleton Microfilament Proteins,tubulin and fibrous protein.