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Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Texas A&M University

Delivery of macromolecules into live cells by simple co-incubation with a peptide.

We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).

Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.

Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).

Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.

Cell-penetrating peptides such as TAT or R9 labeled with small organic fluorophores can lyse endosomes upon light irradiation. The photoendosomolytic activity of these compounds can in turn be used to deliver proteins and nucleic acids to the cytosol of live cells with spatial and temporal control. In this report, we examine the mechanisms by which such fluorescent peptides exert a photolytic activity using red blood cells as a membrane model. We show that the peptides TAT and R9 labeled with tetramethylrhodamine photolyze red blood cells by promoting the formation of singlet oxygen in the vicinity of the cells' membranes. In addition, unlabeled TAT and R9 accelerate the photolytic activity of the membrane-bound photosensitizer Rose bengal in trans, suggesting that the cell-penetrating peptides participate in the destabilization of photo-oxidized membranes. Peptides and singlet oxygen generators therefore act in synergy to destroy membranes upon irradiation.

Synergy between cell-penetrating peptides and singlet oxygen generators leads to efficient photolysis of membranes.

Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide.

Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed.

Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges.

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Article	Year
Delivery of macromolecules into live cells by simple co-incubation with a peptide. Chembiochem : a European journal of chemical biology\| PubMed ID: 20029930	2010
Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans. Bioconjugate chemistry\| PubMed ID: 21043514	2010
Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine. PloS one\| PubMed ID: 21423812	2011
Synergy between cell-penetrating peptides and singlet oxygen generators leads to efficient photolysis of membranes. Photochemistry and photobiology\| PubMed ID: 23278754	2013
Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide. Biochimica et biophysica acta\| PubMed ID: 24135456	2014
Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 24223492	2012
Protein delivery into live cells by incubation with an endosomolytic agent. Nature methods\| PubMed ID: 24930129	2014

Alfredo Erazo-Oliveras

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