中药物质资源开发是一个重要研究领域，长期以来中药化学或现代药理学研究均侧重于中药次生代谢产物，而中药多糖在相关研究中往往被当作杂质除去。随着糖生物学的发展，越来越多的研究表明中药多糖具有很强的生物活性，针对其开发药物递送系统用于解决现有疾病治疗中的问题具有独特的优势。传统中药黄芪一直被认为是改善人体免疫系统的良药，已有报道黄芪多糖具有增强机体适应性免疫的作用，黄芪多糖结构的可修饰性和良好的生物相容性使其在药物递送领域中显示出广阔的应用前景。因此，本研究基于“药辅合一”的理念，以中医肿瘤临床最为常用的补气药黄芪为切入点，从不同来源黄芪多糖的构效关系研究出发，阐明其差异性并以筛选的优效多糖作为新型递送载体，开发系列制剂用于增强抗肿瘤免疫反应，为肿瘤免疫治疗和新药开发提供新的策略和选择。   

目的：目前对于黄芪多糖的研究存在一个关键问题，即受药材来源、提取方法等影响，黄芪多糖的分子结构尚不确定，结构与药效关系并不明确。因此，本研究旨在（1）对仿野生和育苗移栽两种市场上流通黄芪的糖类成分进行分离纯化和结构表征，阐明黄芪多糖的物质基础，为建立黄芪多糖的质量评价标准提供实验依据；（2）对纯化多糖进行活性筛选及初步药效学评价；（3）进一步围绕活性良好的黄芪多糖，构建多糖-肿瘤抗原纳米疫苗，评价其对黑色素瘤和肺癌小鼠肿瘤模型的药效及机制；（4）基于化疗药物诱导免疫原性细胞死亡联合黄芪多糖发挥佐剂活性的设计思路，构建基于黄芪多糖的自聚集纳米胶束，避免无效材料的引入，产生多靶点联合的治疗效果。全文以期对黄芪道地药材的保护、发展和传统中药药效保证提供科学基础和研究思路，为免疫治疗提供新的候选制剂。                               

方法：（1）结构鉴定：首先，我们收集了4年生仿野生芪和2年生育苗移栽芪，通过水提醇沉和色谱分离纯化技术，分别得到w-APS和c-APS两种多糖。采用高效凝胶渗透法（GPC）、高效液相色谱（HPLC）、紫外光谱法（UV）、红外光谱法（FT-IR）对w-APS和c-APS的分子量、单糖组成、纯度、特征官能团等初级结构进行鉴定。通过扫描电镜（SEM）、原子力显微镜（AFM）、刚果红实验和分子模拟，对w-APS和c-APS的高级结构进行表征。（2）黄芪多糖的免疫活性筛选及初步药效学评价：通过CCK-8法测量APS对DC、淋巴细胞活力的影响；采用流式细胞术，对其促进DC表面成熟标志物的表达进行测定，对w-APS和c-APS促进DC成熟的分子机制进行考察；通过APS与免疫抑制剂环磷酰胺联合治疗小鼠乳腺癌，初步评价两种黄芪多糖体内药效。（3）黄芪多糖纳米疫苗的制备及抗肿瘤评价：采用微流控法制备 APS-OVA一体化纳米疫苗，评价其粒径、PDI、稳定性和体外释放行为等性质；采用免疫正常小鼠黑色素瘤、肺癌模型和免疫缺陷小鼠黑色素瘤模型，考察 APS-NVs 和 APS(HMw)-NVs 的抗肿瘤免疫药效及作用机制。（4）基于黄芪多糖的纳米递送系统的构及评价：将疏水性药物硼替佐米（抑制核因子NF-κB）通过硼酯键与APS连接，形成两亲性聚合物，采用探头超声法使DOX进入疏水空腔，制得酸敏感性载药胶束 APS-BTZ@DOX ，并对该多功能纳米粒子进行制剂学和抗肿瘤药效评价，探究黄芪多糖协同ICD诱导剂的药效机制。

结果：（1）结构鉴定：w-APS（1.19×10⁵ Da）和c-APS（1.35×10⁶ Da）主要由葡萄糖（≥85%）、半乳糖和阿拉伯糖组成，比例相似。w-APS和c-APS分别由1,4-α-Glcp、1,2-α-Glcp和1,4,6-α-Glcp按不同比例组成，且w-APS的侧链比例高于c-APS。刚果红染色显示两种黄芪多糖均具有三螺旋结构。此外，w-APS表现出纳米级的球形结构（252.33 nm），而c-APS表现出微型的纤维状结构（743.60 nm）。上述结果表明不同来源的黄芪多糖在结构上存在明显差别。（2）黄芪多糖的免疫活性筛选及初步药效学评价：细胞毒性实验表明，两种来源的黄芪多糖在32-500 μg/mL的浓度下均对DC和淋巴细胞没有杀伤作用;培养12小时后，w-APS和c-APS都能明显诱导CD80、CD86和MHC-II的上调表达，BMDC中共刺激分子的表达与w-APS呈明显的剂量依赖关系，表明w-APS具有促进DCs的增强成熟的能力；与c-APS相比，w-APS在肿瘤组织中的荧光强度明显增加，并且观察到更长的保留时间。抗肿瘤研究表明，w-APS单独或与环磷酰胺联合治疗可明显抑制肿瘤生长，并通过促进免疫抑制细胞和细胞因子的产生而诱发抗肿瘤免疫反应。上述结果表明，w-APS比c-APS表现出更强的抗肿瘤免疫活性及辅助治疗功能，具体机制可能涉及对免疫细胞调控强度不同和体内转运行为有关。（3）制备黄芪多糖纳米疫苗及抗肿瘤评价：采用微流控法制备APS-OVA一体化纳米疫苗，APS作为药物载体和免疫佐剂用于有效的肿瘤免疫治疗。APS-NVs约160 nm，粒径分布均匀，在生理盐水溶液中稳定性良好。摄取实验表明，APS-NVs中的FITC-OVA可以被DCs有效摄取，刺激DCs的成熟，提高体外抗原交叉呈递效率，其机制可能是APS通过TLR4受体诱导DCs激活。皮下注射后，APS-NVs在引流淋巴结和远端淋巴结中的积累都得到了加强，这表明相较于粒径较大的APS（HMw）-NVs，APS-NVs更容易地进入淋巴结，明显提高抗原对DCs的传递效率并激活细胞毒性T细胞；此外，与OVA+AL阳性对照组相比，APS-NVs在B16-OVA黑色素瘤模型中取得了更好的抗肿瘤效果，可能与肿瘤组织的细胞毒性T细胞的增加有关；APS-NVs在黑色素瘤裸鼠模型中抑制肿瘤的效果不佳，也侧面证明了纳米疫苗所诱导的抗肿瘤特异性免疫反应的参与。最后，从小鼠Lewis肿瘤中提取出肿瘤特异性抗原，结合黄芪多糖自组装制备了针对Lewis肺癌的肿瘤特异性疫苗。研究结果表明，制备的新型肿瘤疫苗同样具有良好的抗肿瘤免疫应答反应。（4）基于黄芪多糖的纳米递送系统的构建及评价：所合成的天然多糖键合药胶束APS-BTZ@DOX经体循环进入肿瘤细胞，在肿瘤组织低pH的环境下释放化疗药物杀伤肿瘤细胞，促进肿瘤抗原的产生。APS具有靶向迁移至淋巴结的能力，通过激活DC细胞促进抗原呈递从而诱发更加强大的淋巴细胞杀伤作用，达到药物协同治疗的目的。APS-BTZ@DOX在小鼠黑色素瘤模型中，显示出了良好的治疗效果。

结论：本论文系统比较了仿野生和育苗两种不同来源的黄芪中纯化多糖的结构差异。通过体内外免疫活性评价其对DC、淋巴细胞、巨噬细胞免疫调控功能的影响，发现w-APS可通过与DC细胞的TLR4受体作用，促进DC的成熟，增强其抗原呈递能力。采用微流控技术制备不同栽培方式黄芪的多糖纳米疫苗，发现以w-APS作为递送载体的纳米粒在诱导机体产生特异性免疫反应方面效果更好，可以引起CD8+ T细胞应答，进而激活抗原交叉途径。综上，w-APS为同时具备“药”属性与“辅”属性的黄芪多糖组分，进一步选用w-APS作为载体递送小分子化疗药物，得到具有良好肿瘤治疗效果的pH响应纳米胶束。本研究为黄芪多糖的品质控制和基于黄芪多糖的“药辅合一”纳米递药系统在肿瘤免疫中的应用提供了实验依据。

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 eval

uation 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 B16-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.