1. Assessing the Translocation of CPPs using Force Measurement, Single Molecules and Emulsions
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Nicolas Rodriguez, Sandrine Sagan, Alain Joliot, Sophie Cribier, Pierre Soule, Abdou Rachid Thiam, Frederic Pincet, Université Pierre et Marie Curie - Paris 6 (UPMC), Yale University School of Medicine, Laboratoire de Physique Statistique de l'ENS (LPS), Université Paris Diderot - Paris 7 (UPD7)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Analyse, Interactions Moléculaires et Cellulaires (LBM-E2), Laboratoire des biomolécules (LBM UMR 7203), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC), Yale School of Medicine [New Haven, Connecticut] (YSM), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Département de Chimie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
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0303 health sciences ,Total internal reflection fluorescence microscope ,Chemistry ,Bilayer ,Electroporation ,Biophysics ,Biological membrane ,Cell membrane ,03 medical and health sciences ,Cytosol ,0302 clinical medicine ,Membrane ,medicine.anatomical_structure ,Biochemistry ,medicine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Lipid bilayer ,030217 neurology & neurosurgery ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology - Abstract
Gene therapy and inhibition of malign molecules inside abnormal cells rely on an efficient and specific delivery of drugs into the targeted cells. To do so, disruptive methods such as electroporation or partial destruction of the cell membrane through chemical reagent are currently used.An alternative technique is the use of carriers that will help the drugs to cross the membrane, without introducing deleterious effects due to the membrane disruption. A family of such carriers is known as Cell Penetrating Peptides (CPPs). Those peptides are short, about ten amino acids, and often cationic. They are able to translocate through the membrane with different cargos and deliver them into the cytosol. However the mechanisms are still unknown.We use three different techniques to gain insights into the mechanism leading to the translocation of a CPP. One method use a force measuring tool adapted to living cells: the Biomembrane Force Probe. It gives us the partners that can be involved in such process. A second technique is based on a TIRF microscope where the reflection is made on a suspended bilayer due to a high optical index differences between each side of the membrane. This allows us to follow single peptides close to a suspended bilayer. We managed to observe single CPPs that are in suspension or bound to the membrane. A third method relies on a water-oil emulsion. Two water droplets, one containing fluorescent CPPs and the other only buffer, are generated in an oil-lipid mixture, so that a lipid bilayer is formed at their interface. We follow the translocation of the fluorescent CPPs between the two droplets.So far we were able to identify sugars as binding partner of the CPPs and to image CPP translocation.
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