A GFP Complementation System for Monitoring and Directing Nanomaterial Mediated Protein Delivery to Human Cellular Organelles
A GFP Complementation System for Monitoring and Directing Nanomaterial Mediated Protein Delivery to Human Cellular Organelles
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DOI：
10.1002/bit.22897
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发表时间：
2010-12-15
影响因子：
3.8
通讯作者：
Dordick, Jonathan S.
中科院分区：
文献类型：
--
作者：
Bale, Shyam Sundhar;Kwon, Seok Joon;Dordick, Jonathan S.
基于蛋白质的疗法因其生物相容性和针对特定靶点的活性而变得越来越重要。当这些靶标位于细胞内时，将生物分子递送至细胞质中的位点同时在复杂的细胞环境中保留生物分子活性至关重要。然而，除非伴随着主动摄取机制或载体介导的递送，否则细胞内蛋白质递送是不可行的。此外，一旦进入细胞，生物分子的检测需要费力的技术，缺乏实时测量。我们开发了一种基于荧光的互补蛋白递送传感系统，使用分裂的绿色荧光蛋白（GFP（1-10）和GFP（11））片段，该系统可用作蛋白质递送和活性保留的指示剂，并作为精确定位亚细胞定位的手段。我们通过将 GFP(11) 片段表达到人类细胞的线粒体外膜上，并使用模型载体（15 nm 二氧化硅纳米粒子）来递送 GFP(1-10) 以及图像传输和蛋白质递送的线粒体定位来演示体外局部递送。我们的结果表明，纳米级材料可以用作蛋白质载体，用于靶向细胞成分，包括功能分子、信号通路和细胞器。我们设想，这种 GFP 互补系统非常适合引导基于纳米颗粒的药物和其他生物活性分子递送到细胞内的亚细胞位置，这可以在高通量筛选方案的背景下影响蛋白质-蛋白质相互作用、信号转导途径和体外细胞器功能。生物技术。生物工程。 2010；107：1040-1047。 (C) 2010 年 Wiley 期刊公司。
Protein-based therapeutics are gaining importance for their biocompatibility and activity toward specific targets. When these targets are intracellular, it is critical to deliver biomolecules to sites in the cell cytoplasm while retaining biomolecule activity in the complex cellular milieu. However, intracellular protein delivery is not viable unless accompanied by an active uptake mechanism or carrier mediated delivery. Moreover, once entry into the cell is achieved, detection of the biomolecule requires laborious techniques that lack real-time measurement. We have developed a fluorescence-based complementary protein delivery sensing system using split green fluorescence protein (GFP(1-10) and GFP(11)) fragments, which can be used as an indicator for protein delivery and retention of activity, and as a means to pinpoint subcellular localization. We demonstrate in vitro localized delivery by expressing the GFP(11) fragment onto the mitochondrial outer membrane of human cells, and using a model carrier (15 nm silica nanoparticles) to deliver GFP(1-10) and image trafficking and mitochondrial localization of protein delivery. Our results indicate that nanoscale materials can be used as protein carriers for targeting cell constituents including functional molecules, signaling pathways, and organelles. We envision that this GFP complementation system is ideally suited for directing nanoparticle-based delivery of drugs and other bioactive molecules into subcellular locations within cells, which can impact protein-protein interactions, signal transduction pathways, and organelle function in vitro within the context of high-throughput screening protocols. Biotechnol. Bioeng. 2010;107: 1040-1047. (C) 2010 Wiley Periodicals, Inc.