CATIONIC LIPID PHASE BEHAVIOR AND ITS IMPORTANCE TO TRANSFECTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。重要的治疗程序，如基因转染和基因沉默，需要有效地将遗传物质输送到细胞中。合成阳离子类脂与多阴离子DNA形成复合物（脂丛），是很有前途的基因载体。了解脂质介导的DNA传递机制（脂质感染）对于其成功应用以及合理设计和合成新型阳离子类脂化合物以增强基因传递至关重要。合成了两种具有不对称烃链的新型阳离子磷脂衍生物：油基癸烷酰-乙基磷脂酰胆碱（C18:1/C10-EPC）和硬脂酰癸烷酰-乙基磷脂酰胆碱（C18:0/C10-EPC）。前者作为DNA转染剂（HUAEC细胞）的效果是后者的50倍，尽管它们的化学结构相似，脂质体组织几乎相同。C18:1/C10-EPC相对于C18:0/C10-EPC（以及许多其他阳离子类脂）更有效的一个可能原因是，当这些类脂与带负电的膜脂制剂混合时，所进化的相。通过小角x射线衍射观察，饱和的C18:0/C10-EPC在与生物膜模拟脂质配方（MM: DOPC/DOPE/DOPS/Chol 45:20 20:15, w/w）的混合物中保持了其片层结构；相比之下，不饱和C18:1/C10-EPC在这种混合物中表现出片层-非片层相变。具体来说，在C18:1/C10-EPC与MM和肝脂提取物的混合物中，在~37℃的生理温度下，相转变为非层状相（分别为立方Pn3m和六方HII相）。这些结果强调了载体脂质/细胞脂质混合物的相位特性是转染成功的决定性因素，并为合理设计优质阳离子脂质载体提出了一种新的策略。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Important therapeutic procedures, such as gene transfection and gene silencing, require efficient delivery of genetic material to cells. Synthetic cationic lipoids, which form complexes (lipoplexes) with polyanionic DNA, are promising gene carriers. Understanding the mechanism of lipid-mediated DNA delivery (lipofection) is essential for their successful application, as well as for rational design and synthesis of novel cationic lipoid compounds for enhanced gene delivery. Two novel cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: oleoyldecanoyl-ethyl-phosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). The former was 50x more effective as a DNA transfection agent (HUAEC cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1/C10-EPC relative to C18:0/C10-EPC (and to many other cationic lipoids, as well) was suggested by the phases that evolved when these lipoids were mixed with negatively-charged membrane lipid formulations. As observed by using small-angle X-ray diffraction, the saturated C18:0/C10-EPC preserved its lamellar structure in mixtures with a biomembrane-mimicking lipid formulation (MM: DOPC/DOPE/DOPS/Chol 45:20:20:15, w/w); in contrast, the unsaturated C18:1/C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures. Specifically, in mixtures of C18:1/C10-EPC with MM and liver lipid extract, phase transitions to nonlamellar phases (cubic Pn3m and hexagonal HII phase, respectively) took place at physiological temperatures, ~37¿C. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for transfection success, and suggest a novel strategy for the rational design of superior cationic lipid carriers.