Functionalized Lipid Carriers for Nucleic-Acid and Drug Therapeutics

项目概要/摘要 目前的研究活动水平涉及基因治疗的合成载体（载体）或工程病毒 是史无前例的脂质体是核酸和药物的非病毒载体， 交付应用程序。阳离子脂质体（CL）是目前临床试验中使用的相对安全的非病毒载体。CLS 可以通过静电相互作用与治疗性NA（阴离子DNA或短干扰RNA）复合， 基因递送和沉默，或用作包封在其脂质中的强效细胞毒性疏水药物的载体 双层，在癌症治疗中。在非病毒载体的最大优点中（相对于目前被称为病毒载体的病毒载体）， 在体内环境中更有效）是它们的安全性、它们的低免疫原性和它们转移整个基因的能力 （含有编码和非编码序列）和调控序列进入细胞（目前用工程化的 病毒，因为衣壳大小限制）。具有药效竞争性的非病毒脂质载体的开发 将需要从机理上理解合成载体如何被功能化， 克服内体逃逸的主要细胞内障碍。成功的内体逃逸是释放 治疗性核酸在细胞胞质溶胶内，因此具有最大功效。本研究申请的第一个目的是 采用现代生物物理和合成方法来合理设计功能化的CL-NA纳米颗粒 (NPs)具有协同、互补的双功能PEG-脂质和融合成分， 逃跑本文将利用现代有机化学和固相化学方法合成具有双功能的聚乙二醇脂质体 具有细胞靶向和内体逃逸特性。本研究应用的第二个目的是优化疗效 一种新型的基于CL的疏水药物紫杉醇（PTXL）载体，用于癌症治疗。这将是 通过发展一个机械的理解之间的关系的物理和化学性质的 载体（即官能化CL载体的尺寸、膜自发曲率和脂质尾部结构）和官能化CL载体的尺寸 效力（即PTXL膜溶解度、载体的细胞摄取和PTXL递送导致对人的细胞毒性） 癌细胞）。基于CL的NA和疏水性药物载体的结构将使用低温技术表征。 电子显微镜和同步加速器X射线衍射技术。CL载体与细胞的相互作用 用旋转圆盘共聚焦荧光显微镜直接观察细胞器。他们的结构将是 与它们在人类癌细胞中的生物活性相关。我们研究的广泛而长期的目标是开发一种 基础科学基础，通过机制研究，将导致非病毒载体的设计和合成， 用于基因和癌症治疗的核酸和疏水药物。

Project Summary/Abstract The current level of research activity involving gene therapy with either synthetic vectors (carriers) or engineered viruses is unprecedented. Liposomes are the most widely studied nonviral carriers worldwide for nucleic acid (NA) and drug delivery applications. Cationic liposomes (CLs) are relatively safe nonviral vectors used in ongoing clinical trials. CLs may either be complexed via electrostatic interactions with therapeutic NAs (anionic DNA or short interfering RNA) for gene delivery and silencing, or used as vectors of potent cytotoxic hydrophobic drugs, encapsulated within their lipid bilayer, in cancer therapeutics. Among the biggest advantages of nonviral vectors (over viral vectors which are currently more efficient in in vivo settings) are their safety, their low immunogenicity and their ability to transfer entire genes (containing coding and noncoding sequences) and regulatory sequences into cells (currently not feasible with engineered viruses because of capsid size limitations). The development of nonviral lipid-based vectors with efficacy competitive with viral vectors in vivo will require a mechanistic understanding of how synthetic vectors may be functionalized to overcome the major intracellular hurdle of endosomal escape. Successful endosomal escape is required for release of therapeutic nucleic acid within the cell cytosol and therefore maximum efficacy. The first aim of this research application is to employ modern biophysical and synthetic approaches to the rational design of functionalized CL–NA nanoparticles (NPs) with synergistic, complementary dual-function PEG-lipid and fusogenic components for optimized endosomal escape. Modern methods of organic and solid phase chemistry will be employed to synthesize dual-function PEG-lipids with cell targeting and endosome escaping properties. The second aim of this research application is to optimize efficacy of a new class of CL-based carriers of the hydrophobic drug paclitaxel (PTXL) for cancer therapeutics. This will be achieved by developing a mechanistic understanding of the relation between physical and chemical properties of the carrier (i.e. size of the functionalized CL carrier, membrane spontaneous curvature, and lipid tail structure) and functional efficacy (i.e. PTXL membrane solubility, cell uptake of vector and PTXL delivery leading to cytotoxicity against human cancer cells). The structures of CL-based vectors of NAs and hydrophobic drugs will be characterized using cryogenic electron microscopy and synchrotron x-ray diffraction techniques. The interactions between CL vectors and cell organelles will be directly visualized with spinning disk confocal fluorescence microscopy. Their structures will be correlated to their biological activity in human cancer cells. The broad, long-term objective of our research is to develop a fundamental science base through mechanistic studies that will lead to the design and synthesis of nonviral vectors of nucleic acids and hydrophobic drugs for gene and cancer therapeutics.