Uniform And Disperse Selenium Nanoparticles Stabilized By Codonopsis Polysaccharide And Their Anti-hepatoma Activities

Objective:Primary carcinoma of liver（PCL）is the seventh most common cancer in the world.The content of selenium（Se）in liver cancer patients is deficient,and sufficient Se intake plays a key role in protecting liver cells and reducing the incidence of liver cancer.However,the safe range of Se that can be used in vivo is very narrow,and it is easy to cause poisoning due to excessive use.Therefore,it is very important to explore and develop safe Se forms to reduce the toxicity of Se.In recent years,research on nano selenium（Se NPs）has attracted much attention.Compared with traditional Se forms,Se NPs have lower toxicity and better bioavailability due to their unique size effect and surface effect.The use of reducing agents to reduce selenite and selenate is the most common method to prepare Se NPs,but the Se NPs prepared by this method often have high activity due to the lack of adjacent coordinating atoms on the surface.It is unstable under low temperature,and it is easy to aggregate to form more toxic elemental Se.Therefore,dispersants are generally used in the preparation of Se NPs to increase the dispersibility of Se NPs.Polysaccharides have hydroxyl groups in their molecular structures,which can combine with Se through intermolecular hydrogen bonds to prevent Se NPs from aggregating,so polysaccharides are often used to disperse Se NPs.Codonopsis pilosula is a commonly used tonic Chinese medicine,and polysaccharide is one of its main pharmacologically active components.Modern research has found that Codonopsis polysaccharides have various pharmacological activities such as antioxidant,anti-inflammation,and anti-stress.At present,there are many studies on the structural characteristics and pharmacological activities of Codonopsis polysaccharides,but there are few reports on the use of Codonopsis polysaccharides to modify Se NPs.Therefore,the use of Codonopsis polysaccharide to modify Se NPs and improve the utilization value of selenium is a relatively innovative research hotspot.In conclusion,in this study,Codonopsis polysaccharide was extracted and purified from the traditional Chinese medicine Codonopsis Codonopsis.After clarifying the structure of Codonopsis polysaccharide,Codonopsis polysaccharide was used as a dispersant,and a chemical reduction method was used to synthesize Codonopsis polysaccharide/nano-selenium complex to solve the problem of easy aggregation of Se NPs.The structure and stability mechanism of Codonopsis polysaccharide/nano-selenium complexes were characterized and explored to provide experimental basis for the synthesis of Codonopsis polysaccharide/nano-selenium complexes.Finally,combined with the pharmacological activities of Se NPs,pharmacological methods were used toexploretheanti-canceractivityofCodonopsis polysaccharide/nano-selenium complex from the perspective of cell proliferation and apoptosis to clarify its mechanism of action,which will provide theoretical and experimental basis for the treatment of liver cancer.Method:1 Extraction,separation and structural characterization of Codonopsis polysaccharides:Codonopsis crude polysaccharide（CP）was obtained by water extraction and alcohol precipitation;the purity of CP was analyzed by elemental analyzer;the sugar content of CP was determined by phenol-sulfuric acid method;CP was further purified by gel column chromatography（HPGPC）to obtain Codonopsis polysaccharide CPW1;The Mp,Mw and Mn of CPW1 were determined by HPGPC;the monosaccharide composition of CPW1 was detected by ion chromatography（IC）;the functional groups of CPW1 were analyzed by Fourier transform infrared spectroscopy（FT-IR）;The linkage type of the glycosidic bonds of CPW1 was detected by（GC-MS）;the chemical structure of CPW1 was analyzed by one-dimensional and two-dimensional nuclear magnetic resonance（NMR）.2 Preparation and structural characterization of Codonopsis polysaccharide/nano-selenium complex（CPW1-Se）:Using CPW1 as stabilizer,different concentrations of Na₂Se O₃as raw material,and ascorbic acid as reducing agent,composite materials（CPW1-Se）were obtained by chemical reduction method,which were denoted as CPW1-Se0.1,CPW1-Se0.2 and CPW1-Se0.4,respectively.The morphologies of CPW1,Se and CPW1-Se were observed by transmission electron microscopy（TEM）and high-resolution transmission electron microscopy（HRTEM）;The FT-IR spectra of CPW1-Se were measured by FT-IR.Dynamic light scattering（DLS）was used to measure the particle size and zeta potential of CPW1-Se;CPW1-Se0.1,CPW1-Se0.2 and CPW1-Se0.4 were placed in room temperature for 15 days,and each group of particles was measured by DLS diameter and Zeta potential;exploring the stabilization mechanism of CPW1-Se0.2 by X-ray photoelectron spectroscopy（XPS）and X-ray diffraction（XRD）;measuring Se in CPW1-Se0.2 by inductively coupled plasma mass spectrometry（ICP-MS）content.3 Anti-HCC activity and mechanism of CPW1-Se:Human liver cancer Hep G2 and Huh-7 cells were selected as the research objects to explore the anti-cancer activity and mechanism of CPW1-Se.The effects of CPW1,Se NPs and CPW1-Se on the viability of human hepatoma cells and human embryonic kidney 293T cells were detected by CCK-8method;The effect of CPW1-Se on the proliferation of hepatoma cells was studied by plate cloning method;The effect of Se on the cell cycle of hepatoma cells and human embryonic kidney 293T cells;Flow cytometry was used to study the effect of Se NPs on the cell cycle of human embryonic kidney293T cells;Western blotting was used to study the effect of CPW1-Se on liver cancer cells and human embryonic kidney 293T cells Effects of cell cycle and expression levels of apoptosis-related proteins.Result:1 The elemental analysis of CP shows that the N content in CP is only 0.265%,which indicates that there is no protein in CP;the results of phenol-sulfuric acid method show that the sugar content in CP is 96.8%;the Mp,Mw and Mn of CPW1 were measured by HPGPC.The calculated molecular weights were5205,5623 and 4424,respectively.The two monosaccharides,glucose and fructose,were detected in CPW1 by IC,and the molar ratio of the two was0.121:0.879;CPW1 was detected by GC-MS as fructan,and its main connection method was→1)-Fruf-(2→（93.24%）;Combined with NMR analysis,the structural formula of CPW1 was detected asα-D-Glcp-1→（2-β-D-Fruf-1）n→2-β-D-Fruf（n=35-36）.2 It was observed by TEM and HRTEM that CPW1 appeared as a short worm-like chain in water;in the process of forming Se NPs,if CPW1 was not added,the synthesized Se NPs were prone to interact and aggregate,forming a diameter of 300-500.When the concentration of CPW1 is constant at 5mg/m L,with the increase of Na₂Se O₃concentration,the synthesized Se NPs will be more and more inclined to disperse.The average size increased from54 nm to 79 nm;after placing CPW1-Se0.1,CPW1-Se0.2,and CPW1-Se0.4in room temperature for 15 days,the particle size of CPW1-Se0.4 increased on the 7th day and On the 15th day,the particle sizes of CPW1-Se0.1 and CPW1-Se0.2 remained relatively stable;Compared with the zeta potential measured on the first day,the particle sizes of CPW1-Se0.1 and CPW1 The zeta potential of-Se0.4 decreased,while the zeta potential of CPW1-Se0.2remained relatively stable for 15 days;XPS results showed that Se NPs preferentially interacted with OH groups of polysaccharides to form Se-O bonds;XRD results showed that there were significant differences in the XRD spectra of CPW1-Se and CPW1;No lattice planes were displayed in the HRTEM image of CPW1-Se,and no diffraction rings were displayed in the SAED pattern.The above results indicated that Se NPs were fused with polysaccharides to finally obtain amorphous CPW1-Se.3 The results of CCK-8 showed that CPW1 had basically no toxicity to human hepatoma Hep G2,Huh-7 and human embryonic kidney 293T cells;When the concentration of Se NPs was less than 20μg/m L,it had no effect on human hepatoma Hep G2,Huh-7 cells and 293T cells;Compared with CPW1 and Se NPs,CPW1-Se has a more significant inhibitory effect on the viability of human hepatoma Hep G2 and Huh-7 cells,and it has promoted the proliferation of 293T cells when the dose is less than 200μg/m L.Plate cloning results showed that CPW1-Se could significantly inhibit the proliferation of hepatoma cells;Cell cycle results showed that CPW1-Se could induce the cell cycle arrest of human hepatoma Hep G2 and Huh-7 cells in S phase by inhibiting the expression of the proliferation-related protein cyclin A2;CPW1-Se can promote the cell cycle of 293T cells by up-regulating the protein expression levels of cyclin A2 and cyclin B1,which might promote cell proliferation;Se NPs have no effect on the cell cycle of 293T cells;Protein immunoassay detection of apoptosis-related proteins showed that:CPW1-Se can reduce the protein expression levels of PARP and Bcl-2 in human hepatoma cells,and increase the protein expression levels of Cleaved Caspase9 and Cleaved Caspase 3,which can promote apoptosis;When CPW1-Se was administered to 293T cells,the protein expression levels of PARP and Bcl-2were significantly increased,and the protein expression levels of Cleaved Caspase 9 and Cleaved Caspase 3 were significantly decreased,which showed the effect of inhibiting apoptosis.Conclusion:1.Codonopsis polysaccharide was extracted and purified by water extraction and alcohol precipitation.The extraction conditions were mild,the cost was low,and the obtained Codonopsis polysaccharide had high purity.After testing,CPW1 is an inulin-type fructan with a structural formula ofα-D-Glcp-1→（2-β-D-Fruf-1）n→2-β-D-Fruf（n=35-36）.2 During the synthesis of Se NPs,the use of CPW1 as a dispersant can well disperse Se NPs.3 CPW1-Se can play an anti-hepatoma effect by inhibiting the cell cycle of human hepatoma cells and promoting the apoptosis of human hepatoma cells,and it can promote the proliferation of normal human cells and inhibit cell apoptosis,with obvious safety.