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有效地将蛋白质递送至活哺乳动物细胞的胞质溶胶的方法。目前的递送系统如细胞穿透肽（CPP）是低效的，因为它们促进广泛的内体截留和其蛋白质货物的降解。这导致实验伪像，使得蛋白质生物传感器的适当成像不切实际。我们建议通过优化CPP选择性破坏内体膜的能力来解决这个问题，以便实现蛋白质从内体更有效地释放到胞质溶胶中并减少降解。我们的具体目标是：1）鉴定最佳CPP介导的蛋白质递送所需的条件，2）评估和优化设计用于在内体内腔酸化时有效破坏膜的新型CPP系统，3）定义内体pH、CPP浓度和CPP的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测量传递到活细胞中的蛋白质探针的内吞和胞质分布。还将开发可以报告含有CPP-蛋白质缀合物的内体的性质的新型蛋白质探针。我们期望我们的研究结果将提供关键的化学见解的关键步骤内体破坏，并奠定了坚实的基础，合理设计的有效的传递系统，可以实现细胞溶质靶向的蛋白质生物传感器与低背景的非靶向蛋白质。这将不仅使活细胞的显微镜与外部管理的成像探针，但应该有一个重要的影响，在整个领域的交付细胞不可渗透的大分子一般。公共卫生相关性：目前的递送系统在将外部施用的成像探针靶向活细胞方面效率低下，因此，通常不可能用合成大分子对重要的细胞过程进行成像。我们建议基于关键化学见解开发新型和优化的递送系统，以克服这些限制。这项研究不仅在显微镜领域，而且在将细胞不可渗透的药物或诊断试剂输送到患者体内的背景下也会产生重要影响。

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.