Cationic Phospholipids for Transfection

描述(改编自申请人摘要)：本续期申请为 继续对带正电荷的脂类(三酯)进行卓有成效的研究 天然产物磷脂酰胆碱)作为基因传递载体 细胞。该项目成功地达到了最初的目标，同时 揭示以前未被认识但重要的脂质体介导的方面 基因传递。首次合成了磷脂酰胆碱三酯 时间，并且发现除了 最初提出的将DNA输送到细胞的应用。这些化合物 低毒，孵育后仍保持高效转染剂 在血清中，不需要辅脂，并以适当的速度代谢 在体内用作基因或药物输送载体。化学物质的变化 结构导致衍生品的性质大相径庭。全 液晶版本产生了有效的转染剂，前提是 对处方条件进行了精心优化。的标准制定 原型脂质和DNA的复合体形成了一个多层的三明治 DNA和脂类交替排列的结构。发展中的 通过滴定研究络合物结构的复杂方法 量热法、流式细胞术和几种荧光分光光度方法显示 它的形成过程比人们普遍认为的要复杂得多 而配方程序的变化对结构都有很大影响 以及给定脂类的转染性。因此，要发展到临床应用 对于阳离子脂类，有必要阐明它们之间的关系 处方条件、脂质-DNA络合物结构、体内稳定性 培养条件和转染率。因此，委员会的具体目标是 续期申请包括： 1.以我们在第一代化合物方面的经验为基础 合成数量有限的特定结构的新型阳离子磷脂 增强转染性的特性。2.了解配方条件 影响结构、在血清中的稳定性和靶向修饰的能力， 这些化合物和一组精选的其他重要分子的转染复合体 阳离子脂类。3.将转染物的这些性质与 体内外相关条件下的转染率 (包括将该复合体靶向特定的细胞表面标记)。4.至 扩大协作以促进体内应用程序的开发 在临床上应该变得很重要。

DESCRIPTION(adapted from applicant's abstract): This renewal application is to continue a fruitful investigation of positively-charged lipids (triesters of the natural product, phosphatidyicholine) as vehicles of gene delivery to cells. The project was successful in meeting its original goals as well as revealing previously unrecognized but important aspects of liposome-mediated gene delivery. Phosphatidylcholine triesters were synthesized for the first time and found to have a host of useful properties in addition to the originally proposed application of delivering DNA to cells. These compounds exhibited low toxicity, remained efficient transfection agents after incubation in serum, required no co-lipids, and were metabolized at a rate appropriate for use as a gene or drug delivery vehicle in vivo. Variations in chemical structure led to derivatives with widely different properties. All liquid-crystalline versions generated effective transfection agents, provided formulation conditions were carefully optimized. The standard formulation of the complexes of prototype lipid and DNA generated a multi-decker sandwich structure with DNA and lipid in alternating layers. Development of sophisticated methods of studying the structure of the complex by titration calorimetry, flow cytometry and several spectrofluorometric procedures revealed that its process of formation is more complicated than generally appreciated and variation in formulation procedures greatly influence both the structure and transfection activity of a given lipid. Hence, to progress to clinical uses of cationic lipids, it is essential to elucidate the relationship between formulation conditions, lipid-DNA complex structure, stability under in vivo conditions and transfection efficiency. Accordingly, the specific aims of the renewal application are: 1. To build on our experience with the first generation compounds by synthesizing a limited number of new cationic phospholipids with specific properties to enhance transfection. 2. To understand how formulation conditions affect the structure, stability in serum and ability to modify for targeting, the transfection complexes of these and a select group of other important cationic lipids. 3. To relate those properties of the transfection complexes to transfection efficiency under conditions relevant in vitro and in vivo (including targeting of the complex to specific cell surface markers). 4. To expand collaborations to facilitate development of in vivo applications that should become important clinically.