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Biotechnol Bioeng. 2010 Dec 15;107(6):1040-7. doi: 10.1002/bit.22897.

A GFP complementation system for monitoring and directing nanomaterial mediated protein delivery to human cellular organelles.

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1
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.

Abstract

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.

PMID:
20717973
DOI:
10.1002/bit.22897
[Indexed for MEDLINE]

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