Delivery of protein biosensors across the plasma membrane of live cells

描述(由申请人提供)：我们的长期目标是建立一种方法，将蛋白质有效地输送到活哺乳动物细胞的胞浆中。目前的递送系统，如细胞穿透肽(CPPs)，效率低下，因为它们促进了广泛的内噬体包埋和蛋白质货物的降解。这导致了实验制品的出现，使得蛋白质生物传感器的正确成像变得不切实际。我们建议通过优化CPPs选择性地破坏内体膜的能力来解决这个问题，从而实现更有效地将蛋白质从内体释放到胞浆中，并减少降解。我们的具体目标是：1)确定CPP介导的最佳蛋白质传递所需的条件，2)评估和优化新型CPP系统，该系统设计用于在内吞体腔酸化时有效地破坏膜，3)确定内体pH、CPP浓度和CPP内体释放活性之间的关系。为了实现这些目标，我们将使用最近开发的成像技术来明确地测量送入活细胞的蛋白质探针的胞内和胞浆分布。还将开发新的蛋白质探针，可以报告含有CPP-蛋白质结合物的内涵体的性质。我们期望我们的结果将在内体破坏的关键步骤中提供关键的化学见解，并为合理设计高效的递送系统奠定坚实的基础，从而实现对非靶向蛋白背景较低的蛋白质生物传感器的胞浆靶向。这不仅能够用外部给药的成像探针对活细胞进行显微镜观察，而且通常应该对整个细胞不渗透大分子的输送领域产生重要影响。与公共卫生相关：项目叙述目前的递送系统在将外部给药成像探针定向到活细胞方面效率低下，因此，使用合成大分子对重要的细胞过程进行成像通常是不可能的。我们建议基于关键的化学见解开发新的和优化的输送系统，以克服这些限制。这项研究不仅应该在显微镜领域产生重要影响，而且在将细胞不渗透药物或诊断试剂输送到患者体内方面也应该产生重要影响。

DESCRIPTION (provided by applicant): Our long-term goal is to establish a methodology to efficiently deliver proteins to the cytosol of live mammalian cells. Current delivery systems such as cell-penetrating peptides (CPPs) are inefficient because they promote extensive endosomal entrapment and degradation of their protein cargo. This results in experimental artifacts that render the proper imaging of protein biosensors impractical. We propose to solve this problem by optimizing the ability of CPPs to selectively disrupt endosomal membranes so as to achieve a more efficient release of the protein from endosomes into the cytosol and reduce degradation. Our specific aims are to: 1) identify the conditions required for optimal CPP-mediated protein delivery, 2) evaluate and optimize novel CPP systems designed to efficiently disrupt membranes upon acidification of the lumen of endosomes, 3) define the relations between endosomal pH, CPP concentration and endosomal release activity of CPPs. To achieve these goals, we will use a recently developed imaging technique to unambiguously measure the endocytic and cytosolic distribution of a protein probe delivered into live cells. Novel protein probes that can report on the properties of endosomes containing CPP-protein conjugates will also be developed. We anticipate that our results will provide key chemical insights in the critical step of endosomal disruption and lay a firm foundation for the rational design of efficient delivery systems that can achieve cytosolic targeting of protein biosensors with low background of untargeted protein. This will not only enable the microscopy of live cells with externally administered imaging probes but should have an important impact on the entire field of delivery of cell-impermeable macromolecules in general. PUBLIC HEALTH RELEVANCE: Project Narrative Current delivery systems are inefficient at targeting externally administered imaging probes into live cells and, as a result, the imaging of important cellular processes with synthetic macromolecules is often not possible. We propose to develop novel and optimized delivery systems based on key chemical insights that will overcome these limitations. This research should have an important impact not only in the field of microscopy but in the context of delivery of cell-impermeable drugs or diagnostic reagents into patients as well.