由甾体化合物构建新型功能超分子体系及其基因输送机制相关研究

In this project, we propose to design a series of new steroid-based cationic lipid amphiphiles with specific molecular topology and multiple biological functions,and these lipid amphiphiles will be derived from natural steroidal compounds via multi-steps coupling reactions bearing diverse functional building blocks. These steroid-based cationic lipids will be assembled with genes via new supramolecular assembly strategy toward the application as new functional lipid gene carriers,and their self-assembly in solution will be also examined. The structure-activity relationships between molecular structures of the steroid-based lipids and supramolecular achitectures as well as their gene binding and self-assembly will be investigated.Furthermore, size, shape and morphologies of the steroid-lipids/ gene supramolecular aggregates will be carefully examined in line with their cytotoxicity and in vitro gene transfection efficacy in living cells. Moreover,in order to further shed new lights in gene transfection and its related mechanisms at cellular level, intracellular uptake, endocytosis pathway, cellular trafficking and colocalization of the gene/lipid supramolecular systems will be investigated by a variety of translocation pathway-specific inhibitors and subcellular orgenelle biomarker labeling kits, and the intracelluar fates of synthesized functional lipids and their gene complexes will be explored. It can be anticipated that the successful investigation of this project will lead to establishing a steroid based compound platform toward molecular design and construction of new efficient delivery carriers. In addition, the results of as proposed project may also be possible to pave new approach for utilizing renewable natural-based products as effective building blocks for design and construction of advanced biomaterials for human health care.

该自然科学基金面上项目拟采用天然甾体化合物为特征构筑砌块，通过与功能化分子片段偶联，设计、合成系列具有特定拓扑结构与多种生物功能的新型天然甾体化合物衍生的两亲性阳离子脂质功能分子。进而通过设计可控超分子自组装方法，研究上述功能脂质分子与基因类物质的多组分、多层次、多尺度超分子组装，并进一步对功能脂质分子的溶液自组装性能进行研究。探索该类新型脂质功能分子的分子结构及其超分子自组装与基因复合、细胞输送性能的构效关系。在此基础上，将合成制备所得的新型天然甾体阳离子脂质功能分子作为基因输送载体，研究其分子结构及其超分子组装体的拓扑形貌、尺度等对载体/基因复合物的细胞毒性、基因转染效率的影响规律。进一步运用细胞内生物标记物的共定位、细胞内信号转导通路以及输送通路选择性抑制剂等手段，对制备所得新型天然甾体类阳离子脂质功能分子及其与基因的超分子组装体的活细胞内吞输送通路等相关细胞生物学机制进行研究。

该面上项目拟以天然甾体化合物起始原料，设计合成新型两亲性结构的甾体阳离子功能分子，探索该类阳离子有机功能分子结构及其作为基因载体的性能，研究其在溶液中与基因物质的自组装行为与组装体结构，以及考虑从分子水平上对其进行进一步调控，在生物性能评价方面，进行细胞毒性与生物相容性，以及基因输送性能方面的性能测试。.1..甾体阳离子脂质体的性能在很大程度上取决于甾体的类型，动物固醇的脂质体在理化性能和生物性能上与植物固醇有很大区别，动物固醇脂质体在转染性能方面要优于植物固醇脂质体，但其入胞机制均主要与lipid raft和cavoloae介导的途径相关，观察到了精氨酸头基所带来的核定位性能。.2..发现双脂肪链辅助脂质DOPE的引入使得薯蓣甾醇自组装脂质纳米粒比单纯薯蓣甾醇类脂质具有更好的自组装稳定性能，其SiRNA/pDNA复合纳米颗粒具有更好的细胞内吞和基因输送性能和质粒pDNA细胞内表达能力。有利于后续在SiRNA/pDNA多功能基因载体的应用研究开展。.3..为了发展基因传递-细胞内成像双模态治疗成像一体化体系，构建了Pyrene-polyamine阳离子功能分子多胺药质体，并对其作为基因和药物双载体的构效关系进行了深入探索。发现其超分子组装行为与结构与多胺密切相关，胺链长的增加对细胞毒性具有一定的降低作用，该系列药质体和其基因复合物均主要在胞内均主要分布在溶酶体内。构效关系的研究将促进具有成像-治疗一体化功能的脂药体的发展。.4..基于阳离子脂质体中的阳离子氨基酸砌块和甾体砌块，合成以天然阳离子氨基酸（赖氨酸和精氨酸等）和生物活性糖分子作为生物分子组装体构建的重要砌块，分子设计并通过RAFT，ATRP等可控高分子化学合成、及分子自组装等手段制备发展了一系列易制备、可调控、具有低毒、高效传输特点的功能高分子pDNA输送载体。