Study On The Preparation And Immune Enhancement Of Selenized Hericium Erinaceus Polysaccharide PLGA Nanoparticles

Selenized Hericium Erinaceus polysaccharide（SHEP）is an organoselenides formed by the combination of Hericium Erinaceus polysaccharide and selenium.SHEP exhibits higher biological activities,such as cell proliferation and immunomodulation,than selenium and Hericium Erinaceus polysaccharide alone and is more easily utilized by the body and has a wide range of applications in immunomodulation and anti-tumor.However,due to its large particle size,it is still not easily absorbed by the body.Poly（Lactic-co-Glycolic Acid）（PLGA）has excellent biocompatibility and degradability and has been widely used as a drug sustained release carrier to protect the drug from degradation and provide its sustained release,and the degradation products are non-toxic.Therefore,in this study,selenized Hericium Erinaceus polysaccharide nanoparticles（Se-HEP-PLGA-NPs）were prepared by encapsulating selenized Hericium Erinaceus polysaccharide with PLGA.Then,the effects of Se-HEP-PLGA-NPs on the immune activity of splenic lymphocytes and the immune effect of Newcastle disease virus（NDV）vaccine as an immune adjuvant were investigated from several perspectives.This study is divided into three parts as follows:1.Preparation and characterization of Se-HEP-PLGA-NPs:This experiment aimed to optimize the process conditions for the preparation of Se-HEP-PLGA-NPs using response surface methodology and to determine its physical and chemical properties.Based on single factor experiments,response surface methodology was used to test the impacts of the ratio of organic phase（O）to internal aqueous phase（W₁）,the ratio of external aqueous phase（W₂）to colostrum（PE）and the concentration of PLGA on the encapsulation of nanoparticles of selenized Hericium Erinaceus polysaccharide.The products obtained under simulated optimal process conditions were examined by transmission electron microscopy,particle size and Zeta potential.In result,the optimal preparation process of Se-HEP-PLGA-NPs were as follows:the ratio of organic phase（O）to internal aqueous phase（W₁）was 4.66,the ratio of external aqueous phase（W₂）to colostrum（PE）was 7.23 and the concentration of PLGA was 21.19 mg/m L.Under these conditions,the encapsulation rate of nanoparticles of selenized Hericium Erinaceus polysaccharide was 90.21±0.01%.Moreover,the measured particle size was 67.86±1.55 nm,the zeta potential was-10.42±1.26 m V,the surface was smooth,and there were obvious boundaries between particles and no adhesion phenomenon.The results demonstrated that the dispersion of selenized Hericium Erinaceus polysaccharide nanoparticles was optimized by response surface methodology was relatively moderated,which laid the foundation for the subsequent experiments.2.Effects of Se-HEP-PLGA-NPs on splenic lymphocytes in chicken:The aim of this experiment was to investigate the effects of Se-HEP-PLGA-NPs on the immune activity of splenic lymphocytes in chicken.Se-HEP-PLGA-NPs were applied to splenic lymphocytes in chicken,and the safe concentrations of Se-HEP-PLGA-NPs on splenic lymphocytes in chicken were determined by MTT,and then five safe concentrations of Se-HEP-PLGA-NPs,0.061～0.976μg/m L,were added to the chicken splenic lymphocytes culture system respectively.The effects of Se-HEP-PLGA-NPs on the proliferation of splenic T lymphocytes and splenic B lymphocytes in chicken were determined and on the secretion of cytokines IFN-γand IL-4 were determined by ELISA.In result,Se-HEP-PLGA-NPs at concentrations of 0.061-0.488μg/m L significantly promoted the proliferation of splenic T lymphocytes and splenic B lymphocytes in chicken compared with the blank control group,and Se-HEP-PLGA-NPs at a concentration of 0.488μg/m L were significantly higher than those of the SHEP group（P<0.05）.Se-HEP-PLGA-NPs showed a significant promotion of IFN-γsecretion at a concentration of 0.976μg/m L and IL-4 secretion at 0.122μg/m L（P<0.05）compared with the Con A group.The results showed that Se-HEP-PLGA-NPs were able to significantly enhance the immune activity of splenic lymphocytes in chicken.3.Immune-enhancing effects of Se-HEP-PLGA-NPs on Newcastle disease vaccine in chicken:This experiment aimed to investigate the effects of Se-HEP-PLGA-NPs on the immune response to NDV vaccine in chicken.Healthy Hetian chickens were routinely reared up to 14 days old,randomly divided into eight groups of 25 chickens each.All the chickens were immunized with Newcastle disease vaccine（La Sota strain）at a dose of 0.05 m L per chicken,except for the blank control group,and immunized twice at 28 days old.At the same time of each immunization,the experimental groups were injected intramuscularly with 1 m L each of SHEP and Se-HEP-PLGA-NPs at three concentrations of low（1 mg/kg）,medium（2 mg/kg）and high（4 mg/kg）,and the PLGA-NPs group was injected intramuscularly with an equal amount of 2mg/kg PLGA,and the blank control group was injected intramuscularly with an equal amount of normal saline once a day for 3 days.Blood samples were collected on 7,14,21,28 and 35 days after the first vaccination to determine the potency of Newcastle disease antibodies,the content of IFN-γand IL-4 in serum,the proliferation of splenic lymphocytes in chicken,as well as the spleen,bursa of Fabricius and thymus were collected for immune organ indices and histological observation,separately.In result,compared with the SHEP group,the medium and high dose groups of Se-HEP-PLGA-NPs significantly increased NDV antibody titers（P<0.05）,the proliferation of splenic T lymphocytes and splenic B lymphocytes in chicken（P<0.05）,and immune organ indices（P<0.05）.The low,medium and high dose groups of Se-HEP-PLGA-NPs significantly increased the secretions of cytokines IFN-γand IL-4（P<0.05）.Meanwhile,the spleen,bursa of Fabricius and thymus in the Se-HEP-PLGA-NPs groups were histologically intact,with clear boundaries at each site and no lesions.The results indicated that Se-HEP-PLGA-NPs were able to improve the immunization of Newcastle disease vaccine.