Delivery of protein biosensors across the plasma membrane of live cells

跨活细胞质膜传递蛋白质生物传感器

• 批准号: 7816946
• 负责人: Jean-Philippe Pellois
• 金额: $ 26.77万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7816946
• 关键词: Biosensor; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Cytosol; Diagnostic Reagent; Endosomes; Fluorescence; Foundations; Goals; Image; Imaging Techniques; Knowledge; Lead; Learning; Life; Lysosomes; Mammalian Cell; Measures; Mediating; Membrane; Methodology; Microscopy; Modeling; Molecular; Molecular Weight; Morphologic artifacts; Nature; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Population; Problem Solving; Process; Property; Proteins; Reagent; Reporting; Research; Signal Transduction; Structure-Activity Relationship; System; Testing; Time; Viral; Work; base; cellular imaging; cytotoxic; cytotoxicity; design; endosome lumen; imaging probe; improved; innovation; insight; macromolecule; nanoparticle; novel; polyhistidine; prevent; protein aminoacid sequence; protein degradation; public health relevance; synthetic protein; tool

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.

描述（由申请人提供）：我们的长期目标是建立一种方法，以有效地将蛋白质传递到活哺乳动物细胞的细胞质。当前的输送系统（例如细胞穿透肽（CPP））效率低下，因为它们促进了其蛋白质货物的广泛内体夹层和降解。这导致实验性伪影，使蛋白质生物传感器的正确成像不切实际。我们建议通过优化CPP选择性破坏内体膜的能力来解决此问题，从而使蛋白质从内体更有效地释放到细胞质中并减少降解。我们的具体目的是：1）确定最佳CPP介导的蛋白递送所需的条件，2）评估和优化新型的CPP系统，旨在在酸化内体液体时有效破坏膜的新型CPP系统，3）定义内体pH，CPP浓度，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。还将开发有关含有CPP蛋白结合物的内体特性的新型蛋白探针。我们预计，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，并为有效递送系统的合理设计奠定了坚定的基础，该设计可以实现无靶向蛋白质背景低的蛋白质生物传感器的胞质靶向。这不仅将使活细胞具有外部给药的成像探针的显微镜检查，而且应该对整个可渗透细胞可渗透的大分子的递送领域产生重要影响。公共卫生相关性：项目叙事当前输送系统效率低下，靶向外部施用的成像探针到活细胞中，因此，通常无法使用合成大分子的重要细胞过程进行成像。我们建议基于将要克服这些局限性的关键化学见解开发新颖和优化的输送系统。这项研究不仅在显微镜领域都应该产生重要的影响，而且在将细胞不受欢迎的药物或诊断试剂输送到患者的背景下也应产生重要影响。