新型pH敏感口服纳米DNA疫苗的研制

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.

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

DNA疫苗是一种很有潜力的乳腺癌辅助生物治疗手段。与其他给药方式相比口服给药具有安全性好、病人服从性高等有点。本研究利用以海藻酸修饰的壳聚糖纳米颗粒（A.C.NPs）为载体，构建由该载体携带Legumain DNA制成口服疫苗。并在小鼠乳腺癌模型中验证了该疫苗的抗肿瘤作用。我们的研究结果表明海藻酸修饰的壳聚糖纳米颗粒在PH<3.0的条件下聚集形成毫米级的聚合物；当PH>3.0时解聚分散成为纳米级的颗粒。用携带有EGFP质粒的A.C.NPs对小鼠进行灌胃，免疫荧光染色及流式细胞仪分析结果均显示，与未经海藻酸修饰的壳聚糖纳米颗粒（C.NPs）相比，三天后小鼠小肠派氏淋巴结显示出明显的EGFP表达。在小鼠4T1原位乳腺癌模型中将荷瘤小鼠分别用不同载体包裹的legumain DNA灌胃，结果显示A.C.NPs载体组携带Legmain DNA组其肿瘤体积明显小于其他对照组。分离脾脏获得脾细胞与4T1细胞共培养显示装载有Legumain DNA的A.C.NPs处理组，其小鼠脾脏活化的细胞毒性T细胞（CD3+/CD8+/CD25+）与PBS对照组相比增加了4.7倍，与C.NPs载体组相比增加了2.3倍。说明A.C.NPs载体能够有效减少所携带的DNA经口服后在胃液中的降解；在小鼠乳腺癌模型中，采用A.C.NPs载体携带Legumain DNA制备的口服疫苗能够增加DNA疫苗的抗肿瘤效果。该研究成果发表在 PLOS ONE. 8( 4 ); 2013. 在完成项目任务的基础上，我们继续深入研究了将legumain作为肿瘤治疗靶点的探索。通过设计并构建基于legumain剪切底物AAN，并以穿膜肽TAT修饰的抗肿瘤药物载体。实验结果显示我们构建的AAN-TAT-lipo，能有效增加药物在肿瘤的富集，并实现杀伤肿瘤和重塑肿瘤微环境的双重作用。该研究成果发表在NATURE COMMUNICATIONS. 5 (4280) 2014. 本课题在四年的研究工作中较好的达到了预期目标，开发了海藻酸修饰的壳聚糖纳米颗粒和基于legumain剪切底物AAN并结合穿膜肽TAT的抗肿瘤药物载体并申请专利。培养博士生4名，硕士生2名，发表标注本课题资助号的SCI论文7篇。