阳离子型基因载体构建-基于pH敏感可逆动态结合与主客体作用的自组装模式研究

The main aim of this proposal is to develop multifunctional cationic DNA/siRNA delivery nanosystems via self-assembly approach by taking advantage of boronic acid-catechol coupling and host-guest chemistry. Specifically, as to the DNA delivery nanosystem, block copolymers composed of polyethylene glycol and polylysine serve as the main chain and, the amino-functionalized cyclodextrin can be spontaneously conjugated to it via the phenylboronic acid ester bond and host-guest interaction according to the established chemistry mechanism. In this way, the self-assembled polycations can not only protect DNA from ingestion but also intracellularly release DNA to take action due to the breakage of phenylboronic acid ester bond triggered by the pH decline within lysosome. This selfassembly approach would favor the regulation of the charge density of the polycation and the content of the pH-sensitive groups by adjusting the feed ratios, thereby providing a simple way to obtain a deep insight into the contribution of the dynamic balance between the capacities for compactly packing DNA extracellularly and effectively releasing DNA intracellularly to the transfection efficiency. Noticibly, by host-guest chemistry, amino-functionalized cyclodextrin can serve as a template to accommodate various functional compounds to establish multifunctional siRNA delivery nanosystems. As the proof-of-concept, we attempt to introduce boronic acid-modified adamantine, DOX, fluorescent molecules and active targeting molecules into one cyclodextrin-based cationic nanosystem, in hopes of developing a targeting and labelling siRNA/drug co-delivery nanosystems for tumor treatment. In the system, phenylboronic acid moieties are responsible for the enhanced cellular uptake because it can specifically bind and recognize certain polysaccharide moieties on the cell surface.

本申请利用苯硼酸与邻苯二酚的pH敏感可逆结合特性和主客体作用，通过自组装模式构建阳离子型DNA和siRNA载体，为构建高效安全DNA/siRNA载体体系提供了一种全新的思路。我们首先根据肿瘤部位的特殊生理环境，设计了酸敏感可逆自组装聚阳离子基因传递体系。通过苯硼酸酯键和主客体作用力将氨基修饰的环糊精桥接到聚合物聚乙二醇-聚赖氨酸共聚物侧链。这种模式非常有利于通过控制投料比调节载体材料的电荷密度及pH敏感基团含量，从而提供了一种简易的模型来深入研究细胞外紧密结合DNA能力和细胞内有效释放DNA能力的动态平衡关系对于转染的实际影响。此外，我们将利用氨基功能化的环糊精主体作为模板，通过模板-模块的形式将各种功能性客体分子"即插即用"组装到同一siRNA载体体系中，这些功能性成分包括：模拟凝集素识别靶向细胞的含苯硼酸的客体分子；直观示踪的客体分子；起协同治疗的目的抗癌药物分子以及主动靶向成分。

化疗手段是目前治疗癌症最有效的手段之一。随着分子生物学发展，基因治疗被认为有可能从分子水平来根治疾病，用于肿瘤等疾病的治疗。然而，无论是化疗还是基因治疗，都需要建立一种高效低毒的运载体系将治疗基因或药物安全有效地运送至靶组织或靶细胞，进而增强疗效和抑制副作用。肿瘤环境刺激响应型纳米药物控释系统，能够选择性的在肿瘤部位定点释放药物，在患处形成局部药物高浓度，达到有效提高疗效并降低给药量的目的。另一方面，生物体的组成结构拥有各自特殊复杂的生物功能，很难通过人工的方法来复制。直接利用天然产物，通过仿生策略有可能赋予材料超乎想象的生物功能模拟。同源肿瘤细胞之间存在较强的粘附作用，这一特性可用于实现肿瘤自靶向。将纳米粒子用肿瘤细胞膜进行表面功能化修饰将成为一种新的肿瘤治疗策略，有望实现肿瘤的个性化治疗本研究利用苯硼酸酯的pH敏感可逆结合特性，通过自组装模式构建各类递送载体，包括基因载体，疏水药物载体等；构建亚细胞器溶酶体响应的高效安全载体体系。这种模式可以通过简单地控制投料比来调节载体材料的性质，提供了一种简易的模型来深入研究细胞外紧密结合基因药物和细胞内有效释放的动态平衡关系对于效能的实际影响。同时，我们利用仿生概念，模拟细胞和病毒的外层结构。实现了传递体系的高效传递或者生物识别，达到了化学手段所无法实现的目的。