Identification of the Polycation Molecular Factors That Promote the Intracellular Trafficking of Polycation/DNA Complexes

促进聚阳离子/DNA 复合物细胞内运输的聚阳离子分子因子的鉴定

• 批准号: 0828574
• 负责人: You-Yeon Won
• 金额: $ 24万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0828574&HistoricalAwards=false
• 关键词: Identification; Polycation; Molecular; Factors; Promote

CBET-0828574WonPolycation-based gene carriers, though promising as safer alternatives to viral gene carriers, have been limited in large part due to their poor biological activities. Future design and development of better polycation gene carriers will be greatly facilitated by an improved understanding of the relationship between the polycation chemistry and performance mechanisms. Using combined state-of-the-art polymer synthesis and biological imaging techniques, the proposed research aims to address the gap that exists in our understanding of how the chemical and molecular properties of polycation carriers influence their biological activities and delivery performances in overcoming the two important cellular-level transport barriers, i.e., endosomal escape and nuclear import. Two associated specific aims will be addressed. First, the PI will investigate how the polycation molecular characteristics (in particular, the pKa of the amine group in the polycation, and the spacing between the amine groups along the polycation chain) impact the pathways and kinetics of the early intracellular trafficking processes (endocytosis and endosome escape); for this purpose, polycations with systematically varying chemical/molecular structures will be designed and synthesized, and the endosomal trafficking processes of the DNA complexes based on these polycations will be examined by confocal microscopy. Second, the PI will study the interrelationship between the exact subcellular location (cytosol or nucleus) of partial/complete DNA dissociation from the polycation complex and the nuclear import/transcription of the delivered DNA; for this study, a chemically cross-linked polycation carrier that is degradable upon exposure to mild UV irradiation will be developed and utilized to precisely control the timing and location of the DNA release. The proposed research will use a unique combination of polymer science and cell biology methodologies, this research will provide useful information toward understanding (i) the exact chemical and molecular factors that promote the timely escape of polycation/DNA complexes from endosomes and (ii) the exact mechanisms by which certain polycations (such as polyethylenimine) so effectively enhance the nuclear localization and release/transcription of the delivered DNA. A precise molecular-level understanding of these polycation chemistry vs. performance relationships will provide a fundamental basis for developing new materials and strategies for vastly improved efficiencies of non-viral gene delivery systems, and will therefore help further vitalize the gene therapy field toward realizing the full potential of the technology in both conventional and emerging areas of its applications. The proposed research will provide integrated training for graduate and undergraduate students in a multidisciplinary, collaborative and intellectually stimulating environment to learn skills necessary for the future generation of chemical and biological engineering researchers. Aspects of the proposed research will be used to enhance curricula in the area of nanomedicine.

基于聚阳离子的基因载体，虽然有望作为病毒基因载体的更安全的替代品，但在很大程度上由于其较差的生物活性而受到限制。通过对聚阳离子化学和性能机制之间关系的更好理解，将极大地促进更好的聚阳离子基因载体的未来设计和开发。使用最先进的聚合物合成和生物成像技术相结合，拟议的研究旨在解决我们对聚阳离子载体的化学和分子性质如何影响其生物活性和递送性能的理解中存在的差距，以克服两个重要的细胞水平转运障碍，即，内体逃逸和核输入。将讨论两个相关的具体目标。首先，PI将研究聚阳离子分子特性（特别是，聚阳离子中胺基的pKa，以及沿着聚阳离子链的胺基沿着之间的间距）影响早期细胞内运输过程的途径和动力学（内吞作用和内体逃逸）;为此，将设计和合成具有系统变化的化学/分子结构的聚阳离子，并通过共聚焦显微镜检查基于这些聚阳离子的DNA复合物的内体运输过程。其次，PI将研究DNA从聚阳离子复合物中部分/完全解离的确切亚细胞位置（细胞溶质或细胞核）与递送DNA的核输入/转录之间的相互关系;对于本研究，将开发一种在暴露于轻度紫外线照射时可降解的化学交联聚阳离子载体，并用于精确控制DNA释放的时间和位置。拟议中的研究将使用聚合物科学和细胞生物学方法的独特组合，这项研究将提供有用的信息，有助于理解（i）促进聚阳离子/DNA复合物及时从内体逃逸的确切化学和分子因素，以及（ii）某些聚阳离子在一些实施方案中，所述载体（例如聚乙烯亚胺）如此有效地增强所递送的DNA的核定位和释放/转录。这些聚阳离子化学与性能关系的精确分子水平的理解将为开发新材料和策略提供基本基础，以大大提高非病毒基因递送系统的效率，因此将有助于进一步振兴基因治疗领域，实现该技术在传统和新兴应用领域的全部潜力。拟议的研究将为研究生和本科生提供综合培训，在多学科，协作和智力刺激的环境中学习未来一代化学和生物工程研究人员所需的技能。拟议研究的各个方面将用于加强纳米医学领域的课程。