title: Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles.
abstract: Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.
authors: Centre de Recherches de Biochimie Macromoléculaire, CRBM-CNRS, UMR-5237, UM1-UM2, Department of Molecular Biophysics and Therapeutics, University of Montpellier, Montpellier, France.
authors: Anna
authors: A
authors: Rydström
authors: Sébastien
authors: S
authors: Deshayes
authors: Karidia
authors: K
authors: Konate
authors: Laurence
authors: L
authors: Crombez
authors: Kärt
authors: K
authors: Padari
authors: Hassan
authors: H
authors: Boukhaddaoui
authors: Gudrun
authors: G
authors: Aldrian
authors: Margus
authors: M
authors: Pooga
authors: Gilles
authors: G
authors: Divita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D002118:Calcium; D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D006497:Heparan Sulfate; D034741:RNA, Small Interfering