| The development of TCM material resources is an important research area.For a long time,studies in TCM chemistry or modern pharmacology have focused on secondary metabolites of TCM,while TCM polysaccharides have often been removed as impurities in relevant studies.With the development of glycobiology,more and more studies have shown that TCM polysaccharides have strong biological activities,and the development of drug delivery systems for them is uniquely advantageous for solving problems in the treatment of existing diseases.The traditional Chinese medicine Astragalus has been considered as a good medicine to improve the immune system of human body,and it has been reported that Astragalus polysaccharide has the effect of enhancing the adaptive immunity of the body,and the modifiable structure and good biocompatibility of Astragalus polysaccharide make it show a promising application in the field of drug delivery.Therefore,based on the concept of "unity of medicine and complement",this study takes Astragalus,the most commonly used qi tonic in TCM oncology clinics,as the starting point to elucidate the differences in the conformational relationships of Astragalus polysaccharides from different sources and to develop a series of formulations for enhancing the anti-tumor immune response using the selected superior polysaccharides as novel delivery materials,which can provide a basis for tumor The study will provide new strategies and options for immunotherapy and new drug development.Objective:There is a key problem in the current research on Astragalus polysaccharides,namely,the molecular structure of Astragalus polysaccharides is uncertain and the relationship between structure and potency is not clear due to the source of the herb and extraction method.Therefore,this study aimed to(1)Isolate and purify and structurally characterize the glycan components of Astragalus membranaceus,both wild-like and nursery transplanted,and establish quality control evaluation criteria for Astragalus polysaccharides;(2)Conduct activity screening and preliminary pharmacodynamic evaluation of purified polysaccharides;(3)Further construct polysaccharide-tumor antigen nanovaccines around active Astragalus polysaccharides and evaluate their efficacy and mechanism against melanoma and lung cancer mouse tumor(4)Constructing self-aggregating nanomicelles of Astragalus polysaccharides in combination with ICD-inducing drugs for the evaluation of enhanced anti-tumor immune response.In order to provide a research basis for the conservation and development of Astragalus tractus and the assurance of the efficacy of traditional Chinese medicine,and to provide new directions for immunotherapy.Methods:(1)Structural identification:Firstly,we collected 4-year-old mock wild Astragalus and 2-year-old nursery transplants of Astragalus,and obtained two polysaccharides,w-APS and c-APS,respectively,by aqueous alcoholic precipitation and chromatographic separation and purification techniques.The primary structures of w-APS and c-APS were identified by high performance gel permeation(GPC),high performance liquid chromatography(HPLC),ultraviolet spectroscopy(UV),and infrared spectroscopy(FT-IR)for molecular weight,monosaccharide composition,purity,and characteristic functional groups.The high-level structures of w-APS and c-APS were characterized by scanning electron microscopy(SEM),atomic force microscopy(AFM),Congo red experiments and molecular simulations.(2)Screening of immune activity and preliminary pharmacodynamic evaluation of Astragalus polysaccharide:The effect of APS on DC and lymphocyte viability was measured by CCK-8 method;the molecular mechanism of w-APS and c-APS in promoting DC maturation was investigated by measuring the expression of DC surface maturation markers using flow cytometry;the combination of APS and immunosuppressant cyclophosphamide was used to treat mouse The preliminary evaluation of the in vivo efficacy of two Astragalus polysaccharides was carried out by combining APS with the immunosuppressant cyclophosphamide in the treatment of mouse breast cancer.(3)Preparation and anti-tumor evaluation of Astragalus polysaccharide nanovaccines:APS-OVA integrated nanovaccines were prepared by microfluidic method,and their properties such as particle size,PDI,stability and in vitro release behavior were evaluated;the anti-tumor immune efficacy of APS-NVs and APS(HMw)-NVs were evaluated by using immune-normal mouse melanoma and lung cancer models and immune-deficient mouse melanoma model.(4)Construction and evaluation of nanodelivery system based on Astragalus polysaccharide:The hydrophobic drug bortezomib(inhibits nuclear factor NF-κB)was attached to APS via boron ester bond to form an amphiphilic polymer,and DOX was made into the hydrophobic cavity by probe ultrasonication to produce acid-sensitive drug delivery micelles APS-BTZ@DOX,and the multifunctional nanoparticles were evaluated for formulation science and antitumor efficacy.To investigate the pharmacodynamic mechanism of Astragalus polysaccharide synergistic ICD inducers.Results:(1)Structural identification:w-APS(1.19 × 105 Da)and c-APS(1.35 × 106 Da)were mainly composed of glucose(≥85%),galactose and arabinose in similar proportions.w-APS and c-APS were composed of 1,4-α-Glcp,1,2-α-Glcp and 1,4,6-α-Glcp in different proportions,respectively,and the side chains of w-APS were higher than those of c-APS.Congo red staining showed that both Astragalus polysaccharides had triple helix structures.In addition,w-APS exhibited a nanoscale spherical structure(252.33 nm),while c-APS exhibited a miniature fibrillar structure(743.60 nm).(2)Screening of immune activity and preliminary pharmacodynamic evaluation of Astragalus polysaccharides:cytotoxicity assays showed that both sources of Astragalus polysaccharides had no killing effect on DCs and lymphocytes at concentrations of 500-32 μg/mL;after 12 h of culture,both w-APS and c-APS significantly induced the up-regulated expression of CD80,CD86 and MHC-II,and the co-stimulatory molecules in BMDC expression showed a significant dose-dependent relationship with w-APS,indicating that w-APS has the ability to promote enhanced maturation of DCs;compared with c-APS,the fluorescence intensity of w-APS was significantly increased in tumor tissues,and a longer retention time was observed.Antitumor studies showed that w-APS alone or in combination with cyclophosphamide significantly inhibited tumor growth and induced antitumor immune responses by promoting the production of immunosuppressive cells and cytokines.w-APS exhibited stronger antitumor activity than c-APS,which may be related to its molecular and spatial structure.(3)Preparation of Astragalus polysaccharide nanovaccine and antitumor evaluation:APS-OVA integrated nanovaccine was prepared by microfluidic method,and APS was used as a drug carrier and immune adjuvant for effective tumor immunotherapy.aPS-NVs were about 160 nm with uniform particle size distribution and good stability in physiological saline solution.Uptake experiments showed that FITC-OVA in APS-NVs could be effectively taken up by DCs,stimulating the maturation of DCs and improving the efficiency of antigen cross-presentation in vitro,probably by the mechanism that APS induces DCs activation through TLR4 receptor.The accumulation of APS-NVs in both draining lymph nodes and distal lymph nodes was enhanced after subcutaneous injection,suggesting that APS-NVs entered lymph nodes more easily compared to APS(HMw)-NVs with larger particle size,which significantly improved the efficiency of antigen-to-DCs delivery and activated cytotoxic T cells;furthermore,compared with OVA+AL-positive controls,APS-NVs in B 16-OVA melanoma model achieved better anti-tumor effects,which might be related to the increase of cytotoxic T cells in tumor tissues;the poor tumor suppression effect of APS-NVs in melanoma nude mouse model also laterally demonstrated the involvement of anti-tumor specific immune response induced by nanovaccines.Finally,tumor-specific antigens were extracted from Lewis tumors in mice,and a tumor-specific vaccine against Lewis lung cancer was prepared by self-assembly of Astragalus polysaccharides in combination.The results showed that the prepared novel tumor vaccine also had a good anti-tumor immune response.(4)Construction and evaluation of nano-delivery system based on Astragalus polysaccharide:The synthesized natural polysaccharide-bonded micelles,APS-BTZ@DOX,enter tumor cells via the body circulation and release chemotherapeutic drugs to kill tumor cells and promote tumor antigen production in the low pH environment of tumor tissues.APS-BTZ@DOX has shown good therapeutic effects in a mouse melanoma model.Conclusion:In this thesis,we systematically compared the structural differences of purified polysaccharides in Astragalus membranaceus from two different sources,mock wild and nursery.Its effects on the immunomodulatory functions of DCs,lymphocytes and macrophages were evaluated by in vitro and in vivo immunoreactivity,and w-APS was found to promote the maturation of DCs and enhance their antigen presentation capacity by interacting with the TLR4 receptor of DC cells.A polysaccharide nanovaccine from Astragalus membranaceus of different cultivation methods was prepared by microfluidic technology,and it was found that nanoparticles with w-APS as delivery vehicle were more effective in inducing specific immune responses in the organism,which could elicit CD8+T cell responses and thus activate the antigen crossover pathway.In summary,w-APS is a component of Astragalus polysaccharide with both "medicines" and "excipients"properties.Further,w-APS was selected as a carrier to encapsulate small molecule chemotherapeutic drugs to obtain pH-responsive nanomicelles with good tumor therapeutic effects.This study provides an experimental basis for the quality control of Astragalus polysaccharide and the application of the"drug-assisted" nano-delivery system based on Astragalus polysaccharide in tumor immunization. |
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