口服DNA疫苗是一种有效的肿瘤免疫治疗方法，但做为DNA疫苗的载体，先前研发的纳米颗粒载体难以携带DNA通过胃酸环境并进入肠道高效表达肿瘤抗原。我们的前期研究表明，海藻酸在酸性条件下可以形成致密的高分子网状结构，起到保护DNA的目的，故此我们提出了pH敏感抗酸纳米颗粒DNA疫苗的制备及优化策略：（1）应用pH敏感材料海藻酸修饰壳聚糖，提高纳米颗粒DNA载体的体内抗酸能力，从而高效的表达肿瘤抗原；（2）海藻酸和壳聚糖属天然多糖，对正常组织的毒副作用很小，且有较强的免疫源性，有利于巨噬细胞及树突状细胞的吞噬；（3）以肿瘤特异性表面分子Legumain为靶向分子，选取4T1小鼠原位乳腺癌为研究模型，体内验证pH敏感抗酸纳米颗粒DNA疫的治疗效果。本项目将对壳聚糖纳米颗粒的表面修饰、体内的表达情况及体内抑癌效果进行系统的探究，为制备新型纳米颗粒DNA疫苗载体及抗肿瘤治疗提供了新思路及平台

Legumain DNA vaccine has been proven to be a potential treatment approach to protect against breast cancer. However, absence of safe and efficient oral delivery system highly restricts its clinical application. Here, we aim to develop a novel oral delivery system for legumain DNA vaccine with better biocompatibility and efficiency. We constructed alginic acid-coated chitosan NPs (A.C.NPs) and tested its anti-acid characteristics in vitro. Compared to chitosan NPs (C.NPs), A.C.NPs significantly protected DNA from degradation in the condition of pH=1.5. Furthermore, size distribution analysis showed that when pH<3.0, A.C.NPs aggregated to form the micrometer scale complex and dispersed to nanoparticle sized about 314 nm with the increased pH value. To visualize DNA expression, mice were treated by gavage with the A.C.NPs carrying EGFP expression plasmid. Significant EGFP expression can be detected in the intestinal Peryer's patches of mice. Our data showed that tumor volume was significantly smaller in ACL and SL groups compared to other groups. Further, we isolated splenocytes from mice and co-cultured with 4T1 cells pre-stimulated with hypoxia, which intrigued the translocation of legumain from cytoplasm to membrane. FACS assay showed that active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) in ACL group increased 4.5 folds compared to PBS group and 2.3 folds compared to CL group respectively. Our study suggests that chitosan NPs modified with alginic acid could be a potential safe and efficient tool for oral delivery plasmid DNA. Alginic acid-coated chitosan NPs loaded with legumain DNA vaccine can effectively improve auto-immune response and protect against breast cancer.