Functionalized Lipid Carriers for Nucleic-Acid and Drug Therapeutics

项目摘要/摘要 目前的研究活动水平，包括使用合成载体(载体)或工程病毒进行基因治疗 是前所未有的。脂质体是全世界研究最广泛的核酸和药物的非病毒载体。 递送应用程序。阳离子脂质体(CLS)是正在进行的临床试验中使用的相对安全的非病毒载体。CLS 可以通过与治疗性NAS(阴离子DNA或短干扰RNA)的静电相互作用来络合 基因传递和沉默，或用作强效细胞毒性疏水药物的载体，包裹在它们的脂质中 双层，在癌症治疗中。非病毒载体的最大优势(相对于目前 在活体环境中更有效)是它们的安全性、低免疫原性和转移整个基因的能力 (包含编码和非编码序列)和调控序列进入细胞(目前工程处理不可行 病毒由于衣壳大小的限制)。具有药效竞争力的非病毒脂质载体的研究进展 体内的病毒载体将需要对合成载体如何被功能化的机械理解 克服内体逃逸的主要细胞内障碍。需要成功的内体逃逸才能释放 细胞胞浆中的治疗性核酸，因此具有最大的疗效。本研究申请的第一个目的是 是使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、互补的双功能聚乙二醇脂和融合成分，可优化内涵体 逃走。利用现代有机和固相化学方法合成双功能聚乙二醇脂 具有细胞靶向性和内小体逃逸特性。这项研究应用的第二个目标是优化疗效 这是一种新型的用于癌症治疗的疏水药物紫杉醇(PTXL)的CL载体。这将是 通过发展对物理和化学特性之间的关系的机械理解来实现 载体(即官能化CL载体的大小、膜自发曲率和脂尾结构)和官能化 功效(即PTXL膜的溶解性、细胞对载体的摄取和PTXL对人的细胞毒作用 癌细胞)。基于CL的Nas和疏水药物载体的结构将用低温表征 电子显微镜和同步辐射x射线衍射技术。化学发光载体与细胞的相互作用 细胞器将用旋转圆盘共聚焦荧光显微镜直接显示。他们的结构将是 与它们在人类癌细胞中的生物活性有关。我们研究的广泛和长期目标是开发一种 通过机制研究奠定基础，这将导致设计和合成非病毒载体 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.