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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microscopic Imaging Study Section (MI)
Program Officer
Deatherage, James F
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Texas A&M University
Schools of Earth Sciences/Natur
College Station
United States
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Najjar, Kristina; Erazo-Oliveras, Alfredo; Pellois, Jean-Philippe (2015) Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT. J Vis Exp :
Muthukrishnan, Nandhini; Donovan, Stephen; Pellois, Jean-Philippe (2014) The photolytic activity of poly-arginine cell penetrating peptides conjugated to carboxy-tetramethylrhodamine is modulated by arginine residue content and fluorophore conjugation site. Photochem Photobiol 90:1034-42
Meerovich, Igor; Muthukrishnan, Nandhini; Johnson, Gregory A et al. (2014) Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide. Biochim Biophys Acta 1840:507-15
Erazo-Oliveras, Alfredo; Najjar, Kristina; Dayani, Laila et al. (2014) Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods 11:861-7
Muthukrishnan, Nandhini; Johnson, Gregory A; Erazo-Oliveras, Alfredo et al. (2013) Synergy between cell-penetrating peptides and singlet oxygen generators leads to efficient photolysis of membranes. Photochem Photobiol 89:625-30
Erazo-Oliveras, Alfredo; Muthukrishnan, Nandhini; Baker, Ryan et al. (2012) Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel) 5:1177-209
Muthukrishnan, Nandhini; Johnson, Gregory A; Lim, Jongdoo et al. (2012) TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells. Biochim Biophys Acta 1820:1734-43
Srinivasan, Divyamani; Muthukrishnan, Nandhini; Johnson, Gregory A et al. (2011) Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine. PLoS One 6:e17732
Lee, Ya-Jung; Johnson, Gregory; Peltier, Grantham C et al. (2011) A HA2-Fusion tag limits the endosomal release of its protein cargo despite causing endosomal lysis. Biochim Biophys Acta 1810:752-8
Lee, Ya-Jung; Johnson, Gregory; Pellois, Jean-Philippe (2010) Modeling of the endosomolytic activity of HA2-TAT peptides with red blood cells and ghosts. Biochemistry 49:7854-66

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