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

CBET-0828574WonPolyation型基因载体虽然有望作为病毒基因载体更安全的替代品，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理之间关系的更深入的理解，将极大地促进未来更好的聚阳离子基因载体的设计和开发。结合最先进的聚合物合成和生物成像技术，这项拟议的研究旨在解决我们在理解聚阳离子载体的化学和分子性质如何影响其生物活性和传递性能方面存在的差距，以克服两个重要的细胞水平的运输障碍，即内体逃逸和核输入。两个相关的具体目标将得到解决。首先，PI将研究聚阳离子分子特征(特别是聚阳离子中胺基团的pKa，以及聚阳离子链上胺基团之间的间距)如何影响早期细胞内转运过程(内吞和内吞逃逸)的途径和动力学；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，并将通过共聚焦显微镜检查基于这些聚阳离子的DNA复合体的内体转运过程。其次，PI将研究部分/全部DNA从聚阳离子复合体中解离的确切亚细胞位置(胞浆或细胞核)与所交付DNA的核输入/转录之间的相互关系；在本研究中，将开发一种可在温和紫外线照射下降解的化学交联型聚阳离子载体，并利用其精确控制DNA释放的时间和位置。这项拟议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，以了解(I)促进聚阳离子/DNA复合体及时逃逸的确切化学和分子因素，以及(Ii)某些聚阳离子(如聚乙烯亚胺)有效地增强所传递DNA的核定位和释放/转录的确切机制。在分子水平上准确了解这些聚阳离子化学与性能之间的关系，将为开发新材料和新策略，极大地提高非病毒基因传递系统的效率提供基本基础，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。拟议的研究将在一个多学科、协作和激发智力的环境中为研究生和本科生提供综合培训，以学习下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。

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.