title: Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog.
abstract: L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)μl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
authors.affiliation: INSERM, U836, Grenoble Institute of Neuroscience, LabEx Ion Channels, Science and Therapeutics, Grenoble, France; Université Joseph Fourier, Grenoble, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Tisseyre
authors.affiliation: Université Joseph Fourier, Grenoble, France; INSERM, U1039, Radiopharmaceutiques Biocliniques, Grenoble, France.
authors.forename: Mitra
authors.initials: M
authors.lastname: Ahmadi
authors.affiliation: Université Joseph Fourier, Grenoble, France; INSERM, U1039, Radiopharmaceutiques Biocliniques, Grenoble, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Bacot
authors.affiliation: INSERM, U836, Grenoble Institute of Neuroscience, LabEx Ion Channels, Science and Therapeutics, Grenoble, France; Université Joseph Fourier, Grenoble, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Dardevet
authors.affiliation: Université Joseph Fourier, Grenoble, France; INSERM, U1039, Radiopharmaceutiques Biocliniques, Grenoble, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Perret
authors.affiliation: INSERM, U836, Grenoble Institute of Neuroscience, LabEx Ion Channels, Science and Therapeutics, Grenoble, France; Université Joseph Fourier, Grenoble, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Ronjat
authors.affiliation: Université Joseph Fourier, Grenoble, France; INSERM, U1039, Radiopharmaceutiques Biocliniques, Grenoble, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Fagret
authors.affiliation: Université Joseph Fourier, Grenoble, France; CNRS, UMR5525, TIMC-IMAG, Grenoble, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Usson
authors.affiliation: Université Joseph Fourier, Grenoble, France; INSERM, U1039, Radiopharmaceutiques Biocliniques, Grenoble, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Ghezzi
authors.affiliation: INSERM, U836, Grenoble Institute of Neuroscience, LabEx Ion Channels, Science and Therapeutics, Grenoble, France; Université Joseph Fourier, Grenoble, France; Smartox Biotechnologies, Grenoble, France. Electronic address: michel.dewaard@ujf-grenoble.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: De Waard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D048429:Cell Size; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004337:Drug Carriers; D007457:Iodine Radioisotopes; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000378:metabolism*; D060327:Solid-Phase Synthesis Techniques; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D007457:Iodine Radioisotopes; D012604:Scorpion Venoms; C406406:maurocalcine; D014443:Tyrosine
keywords: Cell penetrating peptide; Drug delivery; Maurocalcine; Quantitative evaluation; Radioiodination