PEI-somes and cationization of protein probes for microimaging of nervous system

描述（由申请人提供）：用于神经系统显微成像的PEI-体和蛋白质探针的阳离子化在该提案中，我们将开发用于将荧光标记的全功能抗体递送到活细胞中的技术和载体。这将使活细胞内的免疫组织化学反应成为可能，消除了对它们的固定和处理的需要。其目的是推进神经系统细胞的显微成像。我们的方法将基于阳离子聚合物聚乙烯亚胺（PEI）。我们将开发一种生产这种阳离子聚合物与荧光标记抗体的高度分散复合物的技术，该复合物将自组装成粒径低于200 nm的液体颗粒（PEI-体）。这些非脂质体、非胶束基颗粒将用于功能性抗体的跨膜递送。应用PEI-体作为活细胞免疫组化荧光标记抗体的跨膜载体，将发展成为一种可销售的产品，以满足神经科学和生物医学研究的需要。虽然合成的阳离子聚合物如PEI是有效的DNA载体，但它们以前没有用作蛋白质载体。在我们的初步工作中，我们能够确保强大的复合物形成和功能蛋白质有效地递送到细胞中。该项目的具体目标是：目标1：开发新的使能技术，用于蛋白质的跨膜运输，通过形成PEI-一些颗粒，利用其可逆阳离子化。演示荧光标记抗体进入活细胞的细胞内递送及其与内部分子靶标的特异性反应，可通过两种方式观察-荧光显微镜和光谱学。测试PEI-抗体复合物的强度、其化学性质和特异性反应性。目的2：建立阳离子化抗体在神经系统细胞实时显微成像中的应用。为了评估和优化荧光标记的抗体的跨膜递送的有效性、它们的细胞内动力学、毒性和特异性反应性，我们的初步数据表明基于聚合物的蛋白质载体优于目前使用的基于肽和基于脂质的蛋白质递送剂的上级品质。我们的项目通过开发一种新的抗体载体，填补了蛋白转运体领域的差距。粒子定向细胞内成像将提供优于目前使用的固定细胞方法的技术优势。它将简化细胞内过程的检测和监测，还将提供一种监测治疗毒性和疗效的方法。改进的成像剂的商业潜力是例外的，因为这些试剂广泛用于神经科学和生物医学研究。公共卫生相关性：拟议项目将导致开发一种新的测定方法，以满足神经科学和生物医学研究的需求。这项技术将推进神经系统细胞的显微成像，并实现细胞过程的活细胞实时成像。

DESCRIPTION (provided by applicant): PEI-somes and cationization of protein probes for microimaging of nervous system In this proposal we will develop a technology and a carrier for the delivery of fluorescently labeled fully functional antibodies into live cells. This will make possible immunohistochemical reactions inside live cells, eliminating the need for their fixation and processing. The objective is to advance microimaging of nervous system cells. Our approach will be based on a cationic polymer polyethyleneimine (PEI). We will develop a technology for the production of highly dispersed complexes of this cationic polymer with fluorescently-labeled antibodies, which will self-assemble into liquid particles with particle sizes below 200 nm (PEI-somes). These non-liposomal, non-micelle-based particles will be used for transmembrane delivery of functional antibodies. Application of PEI-somes as transmembrane carriers of fluorescently labeled antibodies for live cell immunohistochemistry will be developed into a marketable product for the needs of neuroscience and biomedical research. Although synthetic cationic polymers such as PEI are efficient DNA carriers, they were not used before as protein carriers. In our preliminary work we were able to assure strong complex formation and efficient delivery of functional proteins into cells. The specific aims of this project are: Aim 1: To develop the new enabling technology for the transmembrane transportation of proteins using their reversible cationization via formation of PEI-some particles. Demonstrate intracellular delivery of fluorescently labeled antibodies into live cells and their specific reactions with internal molecular targets, observable through two modalities - fluorescent microscopy and spectroscopy. To test the strength of the PEI-antibody complexes, their chemical properties and specific reactivity. Aim 2: To develop an application of cationized antibodies for the real-time microimaging of nervous system cells. To assess and optimize the effectiveness of transmembrane delivery of fluorescently tagged antibodies, their intracellular dynamics, toxicity and specific reactivity our preliminary data indicate superior qualities of the polymer-based protein carriers over the currently used peptide- and lipid-based protein delivery agents. Our project fills the gap in the field of protein transporters by developing a new antibody carrier. The particle-directed intracellular imaging will provide a technological advantage over currently used fixed-cell approaches. It will simplify detection and monitoring of intracellular processes and will also provide a method to monitor both toxicity and efficacy of treatment. The commercial potential for improved imaging agents is exceptional, as these agents are widely used in neuroscience and biomedical research. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of neuroscience and biomedical research. This technology will advance microimaging of nervous system cells and enable live-cell real-time imaging of cellular processes.