| Polysaccharides are a class of natural macromolecules with various bioactivties,including immunoregulatory,antitumor,antioxidant,antiviral,hypoglycemic et.al,among which the antitumor effect is relatively clear.It has been reported that the antitumor activities of polysaccharides are correlated closely to the structure and conformation.The triple helical lentinan,schizophyllan and polysaccharide from Auricularia(A.)auricula-judae exhibit significant antitumor activities,related to the stiff chain conformation in organism and the molecular recognition ability.A.auricula-judae is precious medicinal and edible fungi,and it has been attracted increasing attention due to its multiple biological functions.However,the correlation between structure and antitumor activities of the water-soluble stiff ?-D-glucan from A.auricula-judae is seldom reported.In this thesis,a stiff ?-D-glucan AF1 extracted fromA.aurziculd-judae was successfully fractionated into fractions with relatively low molecular weights by ultrasonic degradation,and the relationship between the structure and anti-hepatoma activities of the stiff?-glucan was revealed.Additionally,the strategy to use AF1 to fabricate well-dispersed Se nanoparticles could be a highly efficient pathway to achieve highly dispersive and stable Se nanoparticles for enhanced safety,biocompatibility and antitumor activities.Therefore,this work is in an interdisciplinary field of polymer chemistry physics,nanomaterials and natural polymers biomedicine,and also the international research frontiers of natural products.The innovation of the thesis are as follows:(I)The stiff>?-D-glucan AF1 isolated from A.auricular-judae was successfully fractionated into fractions with relatively low molecular weights by ultrasonic degradation.The fractionated AF1I glucans with different molecular weights were proved to adopt a stiff chain conformation in water,the same as the unfractionated AF1;(2)The AF1 samples were found to exhibit significant anti-hepatoma activities without cytotoxicity,for the first time.The anti-tumor activity was dependent on the molecular weight and conformation,and the anti-tumor mechanism was also investigated;(3)It has been firstly proposed a green and facile method for fabricating a water-soluble stiff ?-glucan(AF1)entrapped selenium nanoparticles(AFl-Se),and the mechanism was illustrated;(4)AFl-Se exhibited significant anti-breast cancer activities without cytotoxicity,and the mechanism of the apoptosis of human breast cancer MCF-7 cells induced by AF1-Se was investigated;(5)AFl-Se exhibited significant anti-cervical cancer activities without cytotoxicity,and the mechanism of the apoptosis of human cervical cancer Hela cells induced by AFl-Se was investigated.The main contents and conclusions of the thesis are divided into the following sections.The stiff ?-D-glucan AF1 isolated from Auricularia auricular-judae with high molecular weight showed a remarkable dependence of on[n]shear rate.AFI was successfully fractionated into three fractions with different molecular weights by using ultrasonic degradation.The molecular weight and chain conformation of the AF1 samples were successfully characterized by static and dynamic laser light scattering,viscometry and atomic force microscopy.The structure-sensitive parameter(p)values were all greater than 2 in water,revealing the stiff chain conformation of the fractionated and unfractionated AF1 glucans.AFM images directly confirmed AF1 and its fractions existed as stiff chains in water,revealing the ultrasonic treatment only reduced the molecular weight of AF1 R and hardly changed the chain conformation.Moreover,the aggregates appeared with an increase in the glucan concentration,forming a coexistence of individuals and aggregates.Molecular weight and conformation closely related to the anti-tumor activity of bioactive polysaccharide.This work will provide a theoretical basis for studying the molecular weight dependence of its antitumor activity.The AF1 samples were found to exhibit significant anti-hepatoma activities without cytotoxicity,for the first time.Moreover,the anti-tumor activity was dependent on molecular weight and conformation,and the optimal molecular weight of AF1 was estimated to be 7.7 x 105.The inhibition rate of tumor was about 75%,significantly higher than that of positive control cyclophosphamide(62%).It could be explained that the extended chain conformation of AF1 increased the probability of the interaction with receptors,leading to a stronger anti-tumor effect.The anti-tumor activity of AF1 glucans first increased with increasing glucan dosage to reach the optimal dosage of 5mg/kg,and then decreased with further increasing in dosage,as a result of the aggregation of the AF1molecules,which weakened the interaction of glucan with receptors on the cell surface.The underlying anti-tumor mechanism was preliminarily proposed as the AF1 glucan induced cancer cell apoptosis and anti-angiogenesis through activating immune responses in mice.The AF1 ?-glucan was a potent natural drug candidate with high anticancer activity and less cytotoxicity.A green and facile method for fabricating a water-soluble stiff polysaccharide(AF1)entrapped selenium nanoparticles(AFl-Se)has been proposed.The spherical SeNPs with the mean particle size of 46 um were entrapped in the cavities of the AF1 hollow nanofibers through formation of Se-O bands,leading to the highly stable Se nanoparticles.The cavity of the AFl hollow nanofiber effectively stabilized and protected the Se nanoparticles,leading to their good dispersion.The whole process was simple and green.This strategy to use AFl to fabricate well-dispersed Se nanoparticles could be a highly efficient pathway to achieve highly dispersive and stable Se nanoparticles for significantly increasing biocompatibility and safety,possessing potent applications in nanomedical material field.The AF1 hollow nanofibers entrapped SeNPs significantly enhanced the cellular uptake of SeNPs through endocytosis.The AF1-Se nanocomposite exhibited a broad-spectrum inhibition against human cancers,especially breast cancer MCF-7 cells.In vivo animal studies demonstrated that AF1-Se exhibited significant anti-tumor activities without normal tissues toxicity.The results of flow cytometry and western blot demonstrated that cell apoptosis and S phase cell arrest in MCF-7 cells were contribute to the antiproliferative activity of the AF1-Se.Furthermore,the mechanism of apoptosis was the activation of Caspase and ROS accumulation related apoptosis pathway.Thus,the strategy to use AF1 to fabricate well-dispersed Se nanoparticles lead to strong synergy anti-cancer activities,and AF1-Se may be candidates for further evaluation as a potential chemotherapeutic agent for human breast cancer.It has been also revealed that the AFl-Se exhibited higher anti-tumor activities against cervical cancer.The in vitro assay indicated that AF1-Se significant inhibition against Hela cancer cells with IC50 value of 50 ?pg/mL and low toxicity to normal cervical cells H8.Our results demonstrated that cell apoptosis and G2/M phase cell arrest in Hela cells were contribute to the antiproliferative activity of the AF1-Se.Furthermore,AFl-Se induced caspase-dependent apoptosis in Hela cells.The study about signal pathways indicated that AFl-Se activated the intracellular MAPK/P38 signal pathway,inhibiting the MAPK/JNK,MAPK/ERK,PI3K/Akt and STAT3 signal pathways.The in vivo assay indicated that AF1-Se significant inhibition against Hela tumors through inducing cancer cell apoptosis and anti-angiogenesis.The strategy to use AF1 to fabricate well-dispersed Se nanoparticles lead to strong synergy anti-cancer activities.AF1-Se may be candidates for further evaluation as a chemotherapeutic agent for human cervical cancer.This fundamental research focused on correlation of structure to antitumor activities of the stiff ?-D-glucan AFl isolated from A.auricula-judae,and proposed a green method for fabricating AF1 entrapped selenium nanoparticles(AF1-Se).Moreover,the anti-tumor mechanism was also investigated.These results not only displayed scientific significance,but also supplied essential data for the utilization and application of the nanomedical material field. |
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