主-被动协同靶向纳米高分子载体的自组装制备及其抗肿瘤作用机制研究

Clinical reports showed that nano gene medicine such as Allovectin-7 has improved the therapeutic effects. However, its low targeting and low transfection efficiency resluted in unfavourable prognosis. Thus, construction an antibody specifically targeted and high transfection efficient drug delivery system is the key for tumor gene therapy. Previously, we have prepared series of narrow distributed block copolymers by controlled polymerization and sellfassembly.Referring to the Flory parameter (χ) and tuning the composition and packing parameter (β),we investigated the scaling law between the assembly dimension and its composition. The systemically showed that increase temperature enhanced the drug intratumor accumulation with 4 times with obviously enhanced tumor killing. Based on these observation, in this proposal, a Her2 antibody targeted temperature sensitive block copolymer Fab-PNIPAM/PEG/PLA will be further synthesized firstly. Then the SiRNA will be entapped into the vesicle. And the intelligent active/passive cooperative targeting nanocarries will be constructed by the controlled selfassembly technology and referring to the tumor microenvironmen. The virus-like properties including the antibody active targeting, temperature promoted endocytosis, endosomal controlled release will be realized by finely carrier's structural tailoring. And the including mophology, We will systemically investigate the correlations between the selfassembly behavior and carrier's dimension, the carrier's properties, the nucleic acid drug loading efficiency, the active and passive cooperative targeting efficiency, in vitro/vivo gene silencing efficincy. Then we will further reveal the targeting and anti-Her2 over-expression tumor mechnism. The successful accomplish of this project could provid a new idea and theory for clinical gene therapy.

临床证实Allovectin-7等纳米基因药物能有效提高抗肿瘤效果，但靶向性差及转染效率低导致其预后仍不理想。因此，构建抗特异体靶向和高转染效率药物输送体系是基因治疗关键。前期,我们设计合成系列不同嵌段高分子；依据Flory参数（χ）调控嵌段组成及包装因子（β）,研究了组装体维度与组成的标度关系；系统研究发现温敏被动靶向促进药物瘤内富集高达4倍，肿瘤杀伤显著增强。基于上述研究，本课题将进一步针对肿瘤微环境特点，运用可控聚合设计合成抗体Her2偶联温敏篏段聚合物Fab-PNIPAM/PEG/PLA；通过可控自组装技术包裹SiRNA,构建主被动协同靶向泡囊载体；并精细裁剪载体，实现抗体主动靶向、温敏被动靶向及胞内可控释放等性能；系统探讨自组装行为与载体的维度、性能、核酸药物负载率、协同靶向性、体内外基因沉默效率的关系；阐明协同靶向及抗Her2高表达肿瘤机制，为临床基因治疗提供新思路和理论依据。

项目执行期间获得的结果超额完成预定的研究目标和任务。. 本项目重点是围绕纳米医药发展面临的“体外性能好-体内表现差”这一瓶颈问题开展，针对肿瘤是个多基因相关疾病，其组织温度较正常组织偏高等特性，设计构建抗体主动靶向-温度被动靶向等多靶向功能纳米体系，包括纳米泡囊，纳米凝胶、胶束载体，及利用纳米技术组装成多功能具有协同作用机制纳米抗体团簇（Nano antibody, NAb）；并系统研究了各种纳米医药体系的自组装原理、其物理化学性能、体系的主动靶向功能，通过细胞和动物实验研究体系如何通过EPR增强肿瘤富集，系统探讨和阐述了双靶向纳米体系在组织水平、细胞水平的靶向协同、以及基于双靶向功能的协同增强胞内药物作用机制的研究。我们率先提出并进一步证实纳米体系的温度敏感被动靶向功能（Temperature sensitive passive targeting, TSPT）在促进瘤内富集细胞内吞中的效果；其次，我们在前期基础上，进一步提出利用“纳米物理药剂学”原理探讨，来研究纳米体系的物理化学性能，并以此解决纳米医药“体外性能好-体内表现差”这一发展的瓶颈问题。而且，率先利用纳米技术和抗体修饰技术，构建真正的多功能纳米抗体，相比母代抗体，纳米抗体具有EPR增强瘤内富集，降低剂量，多个抗体在肿瘤细胞表面的协同作用增强，并意外诱导凋亡，具有传统抗体不具有的协同抑制肿瘤的能力。. 基于这些研究，我们发表SCI论文11篇（17年高引1篇），中文核心4篇，申请专利4项，授权2项。项目执行期间培养（联合培养）了硕士8名，博士3名，2名硕士获上海市优秀毕业生，一名初级科研助理晋升中级；任《第二军医大学学报》论坛专家组织专栏讨论，在“中青年学者论坛”阐述“纳米物理药剂学”研究的观点。. 基于本课题资助的研究，我们首次系统的获得了系列决定纳米医药体系体内外应用的参数与大分子的组成制剂的相互关系式，设计制备了一套纳米胶束辅料制备的设备，申请专利2项，并与上海医药集团达成900万元受让研发合同。