癌症作为一种易复发、难治愈的疾病严重威胁人类的健康。虽然近年来癌症的治疗方法和手段越来越多样化，但单一的治疗方法往往疗效有限，不足以根除肿瘤。因此联合疗法逐渐被用于癌症的治疗，此时则需要合适的策略实现多种药物的联合递送。以海藻酸钠为基质制备的水凝胶/纳米粒复合体系为肿瘤的化疗联合免疫治疗提供了一种理想的递送平台，可通过化疗药物和免疫佐剂的双持续释放增强体内的抗肿瘤效应，有效抑制肿瘤的生长和转移。

目的：为了克服单一化疗的耐药性和全身毒副作用，以及无法有效抑制肿瘤转移的不足，本文构建了负载药物和纳米粒的海藻酸钠水凝胶复合体系用于黑色素瘤的化疗联合免疫治疗，以期有效抑制近端瘤和远端瘤的生长，预防肿瘤的复发和转移。

方法：我们以稳定性和生物相容性良好的PCL₄₀₀₀-PEG₈₀₀₀-PCL₄₀₀₀为载体材料，利用薄膜水化-超声分散的方法制备了包载疏水性TLR7/8激动剂咪喹莫特（Imiquimod，IMQ）的聚合物胶束，表面用马来酰亚胺功能化磷脂DSPE-PEG₂₀₀₀-Mal、甘露糖靶向磷脂DSPE-PEG₅₀₀₀-Mannose进行修饰并嵌合磷脂类TLR4激动剂单磷酰脂质A（Monophosphoryl lipid A，MPLA），从而制备了具有抗原捕获性能且能程序化递送多组分的脂质聚合物杂化纳米粒（NP-Mal），并对其理化性质和对骨髓来源树突状细胞（Bone marrow-derived dendritic cells，BMDCs）的促成熟作用进行了评估。然后将天然的海藻酸钠、化疗药阿霉素（Doxorubicin，DOX）、IDO抑制剂1-甲基-D-色氨酸（Indoximod，IND）和脂质聚合物杂化纳米粒经物理混合来制备可用于化疗联合免疫治疗的海藻酸钠水凝胶复合体系，并对该体系的理化特性、细胞毒性、生物安全性、储库效应及纳米粒的淋巴结迁移进行了评估。最后，以黑色素瘤为肿瘤模型，评估了该体系抑制近端瘤与远端瘤生长、预防肿瘤转移的效果并对相应的免疫机制进行了研究。

结果：我们成功构建了一种负载药物和纳米粒的海藻酸钠水凝胶复合体系，其中纳米粒（NP-Mal）粒径均一（~165.8 nm），分散性良好，具有较高的IMQ包封率（~83.00%）和良好的抗原捕获能力；负载药物的海藻酸钠水凝胶具有良好的响应钙离子而原位成胶的能力，形成的三维网状立体结构保证了IND和DOX的缓慢持续释放。体外细胞实验表明载药水凝胶（GEL-DOX和GEL-IND-DOX）通过阻滞细胞周期进展和诱导细胞免疫原性死亡对B16F10细胞表现出强大的杀伤作用；而NP-Mal纳米粒具有良好的生物安全性，与肿瘤裂解抗原（Tumor-derived protein antigens，TDPAs）共孵育后能显著促进BMDCs表面CD40、CD86和MHCI的表达与细胞因子TNF-α和IFN-γ的产生。此外，体内实验表明GEL-ID-NP-Mal组显著促进纳米粒的淋巴结迁移及脾内CD4⁺ T细胞、CD8⁺ T细胞、效应记忆T细胞（Effector memory T cells，TEM）的增殖活化和细胞因子TNF-α、IFN-γ、IL-12的分泌；并下调脾内免疫抑制细胞如调节性T细胞（Regulatory T cells，Tregs）和M2型巨噬细胞的比例。同时GEL-ID-NP-Mal有效促进CD8⁺ T细胞和NK细胞浸润到肿瘤组织，并显著下调肿瘤内M2型巨噬细胞的比例。因此，该海藻酸钠水凝胶复合体系（GEL-ID-NP-Mal）通过激活机体的免疫系统，重塑免疫抑制微环境和建立免疫记忆保护来有效抑制原发肿瘤和远端肿瘤的生长及转移，并延长荷瘤小鼠的生存期。

Cancer, as a disease that is easy to recur and difficult to cure, has seriously threaten human health. Although the treatments of cancer have become increasingly diversified in recent years, the therapeutic effect of monotherapy is too limited to completely eradicate tumors. Therefore, combination therapy is gradually used in the treatment of cancer, in which appropriate strategies are needed to achieve the co-delivery of multiple drugs. The hydrogel / nanoparticle composite system prepared with sodium alginate provides an ideal delivery carrier for tumor chemotherapy combined with immunotherapy, which enhances the in vivo anti-tumor immune effect by double continuous release of chemotherapeutic drugs and immune adjuvants, and significantly suppresses tumor growth and metastasis.

Objective: To overcome the drug resistance, systemic toxicity and side effects of single chemotherapy, as well as the inability to effectively inhibit tumor metastasis, this research constructed sodium alginate hydrogel composite system loaded with drugs and nanoparticles for chemotherapy combined with immunotherapy against melanoma, which was expected to effectively inhibit the growth of primary and distal tumors and prevent tumor recurrence and metastasis.

Methods: PCL₄₀₀₀-PEG₈₀₀₀-PCL₄₀₀₀ with good stability and biocompatibility was used to prepare micellar nanoparticles loaded with hydrophobic TLR7/8 agonist IMQ by thin film hydration-ultrasonic dispersion method. Lipid polymer hybrid nanoparticles (NP-Mal) with antigen capture capability and programmed delivery of multi-components were prepared by modifying maleimide and mannose on the surface of the micelles and embedding TLR4 agonist MPLA within the phospholipid layer. The physicochemical properties and effects of NP-Mal on promoting the maturation of bone marrow dendritic cells (BMDCs) were studied. Then, natural sodium alginate, chemotherapeutic drug doxorubicin (DOX), IDO enzyme inhibitor 1-methyl-D-tryptophan (IND) and lipid polymer hybrid nanoparticles NP-Mal were physically mixed to prepare a sodium alginate hydrogel composite system to realize chemotherapy combined with immunotherapy against melanoma. The physicochemical properties, cytotoxicity, biosafety, depot effect and migration to secondary lymphatic organs were evaluated. Finally, B16F10 melanoma was used as the tumor model to evaluate the effect of this hydrogel composite system on inhibiting the growth of primary and distant tumor, preventing tumor recurrence and metastasis and study the relevant immune mechanism.

Results: We successfully fabricated a sodium alginate hydrogel composite system loaded with chemotherapeutic drugs, IDO inhibitor, and antigen-capture nanoparticles (NP-Mal). The NP-Mal exhibited uniform particle size (~165.8 nm) with good monodispersity，high IMQ encapsulation efficiency (~83.00%), and good antigen capture ability. The sodium alginate hydrogel loaded with drugs had the ability to form in situ gel in responsive to calcium ions, and the three-dimensional network structure ensured the slow and continuous release of IND and DOX. In vitro cell study indicated that the drug-loaded hydrogel exhibited a strong killing effect on B16F10 cells by blocking cell cycle progression and inducing immunogenic cell death, and NP-Mal nanoparticles with good biocompatibility significantly promoted the expression of CD40、CD86 and MHC I on BMDCs surface and the production of cytokines TNF-α and IFN-γ after co-incubation with tumor-derived protein antigens. In addition, in vivo experiments showed that the GEL-ID-NP-Mal significantly promoted the migration of the nanoparticles to secondary lymphatic organs, promoted the proliferation and activation of CD4⁺ T cells, CD8⁺ T cells and effector memory T cells (TEM) in spleen, facilitated the secretion of cytokines (TNF-α, IFN-γ and IL-12), and reduced the proportion of immunosuppressive cells such as regulatory T cells (Tregs) and M2 macrophages in spleen. At the same time, it effectively promoted the infiltration of CD8⁺ T cells and NK cells into tumor tissues, and significantly reduced the proportion of M2 macrophages in tumors. Therefore, the sodium alginate hydrogel composite system (GEL-ID-NP-Mal) effectively inhibited the growth of primary and distant tumors, prevented tumor metastasis, and increased survival efficacy of tumor-bearing mice by activating the body's immune system, reshapinng the immunosuppressive tumor microenvironments (TME), and establishing immune memory protection.