title: Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.
abstract: BACKGROUND
  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).
  
  
  RESULTS
  We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.
  
  
  CONCLUSIONS
  Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Divyamani
authors.initials: D
authors.lastname: Srinivasan
authors.forename: Nandhini
authors.initials: N
authors.lastname: Muthukrishnan
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Johnson
authors.forename: Alfredo
authors.initials: A
authors.lastname: Erazo-Oliveras
authors.forename: Jongdoo
authors.initials: J
authors.lastname: Lim
authors.forename: Eric E
authors.initials: EE
authors.lastname: Simanek
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pellois
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002338:Carotenoids / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000528:radiation effects; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004357:Drug Synergism; D004705:Endocytosis / Q000187:drug effects / Q000528:radiation effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D008027:Light; D008969:Molecular Sequence Data; D010778:Photochemotherapy; D010782:Photolysis / Q000187:drug effects / Q000528:radiation effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000528:radiation effects; D012235:Rhodamines / Q000627:therapeutic use*; D026082:Singlet Oxygen / Q000378:metabolism; D014801:Vitamin A / Q000031:analogs & derivatives
chemical_list: C437523:5-carboxytetramethylrhodamine; D057846:Cell-Penetrating Peptides; D012235:Rhodamines; C487773:trans-sodium crocetinate; D014801:Vitamin A; D026082:Singlet Oxygen; D002338:Carotenoids