Direct Translocation as Major Cellular Uptake for CADY Self-Assembling Peptide-Based Nanoparticles

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

A non-covalent peptide-based strategy for siRNA delivery.

International audienceThe development of short-interfering RNA (siRNA) has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of siRNA together with the low permeability of the cell membrane to negatively charged molecules, remain major obstacles to clinical development. So far there is no universal method for siRNA delivery as they all present several limitations. Several non-viral strategies have been proposed to improve the delivery of synthetic siRNAs in both cultured cells and in vivo. Cell-penetrating peptides (CPPs) or protein transduction domains (PTD) constitute very promising tools for non-invasive cellular import of siRNA and non-covalent CPP/PTD-based strategies have been successfully applied for ex vivo and in vivo delivery of therapeutic siRNA molecules. We recently described a new peptide-based system, CADY, for efficient delivery of siRNA in both primary and suspension cell lines. CADY is a secondary amphiphatic peptide able to form stable non-covalent complexes with siRNA and to improve their cellular uptake independently of the endosomal pathway. This chapter describes easy to handle protocols for the use of the CADY-nanoparticle technology for the delivery of siRNA into both adherent and suspension cell lines. It will also highlight different critical points in the peptide/siRNA complex preparation and transfection protocols, in order to obtain siRNA-associated interfering response at low nanomolar concentration

Le TIMP-1 humain, une protéine multifacette (production sous forme recombinante et intérêt thérapeutique)

Les TIMPs (Tissue Inhibitor of Metalloprokinases) sont lès inhihiteurs spécifiques des métalloprokases matricielles (MMPs). Ils participent à la regulation du remodelage matriciel, mais présentent aussi des fonctions indépendantes de cette activité (facteur de croissance, facteur de survie). A travers ces multiples facettes, la protéine TIMP-1 est impliquée dans des processus phvsiologiques et pathologiques. Nos objectifs sont de caractériser et d'utiliser les propriétés biochimiques de TIMP-1 afin d'évaluer son potentiel thérapeutique. Nous avons établi un protocole d'expression en système mammifère puis un schéma de purification de la protéine rh-TIMP-1 (recombinant human TIMP-1) nous permettant d'obtenir 30 mg/l de TlMP-1 pure, dépassant largement les taux dejà décrits. Ceci nous a permis d'évaluer son efficacité en tant qu'agent thérapeutique dans un modèle murin d'arthrite au collagène (mimant la polyarthrite rhumatoïde). Des analvses cliniques et sérologiques suggèrent un effet bénéfique des injections de rh-TIMP-1 à dose élevée contrairement aux doses faibles. Des investigations supplémentaires sont nécessaires pour déterminer le réel effet de TIMP-1. En parallèle, nous avons étudié la propriété de transduction de TIMP-1 dans les cellules tumorales. Nous avons montré que les acides aminés C-terminaux portent cette propriété et désormais nous devons optimiser ce processus et tester le transport d'une protéine thérapeutique (p.53). Ce travail confirme l'intérêt biotechnologique de la proteine TIMP-1 en tant que navette et suggère l'intérêt thérapeutique potentiel qui nécessite des investigations additionnelles en particulier dans d'autres modèles pathologiques.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Le TIMP-1 humain, une protéine multifacette (production sous forme recombinante et intérêt thérapeutique)

Les TIMPs (Tissue Inhibitor of Metalloprokinases) sont lès inhihiteurs spécifiques des métalloprokases matricielles (MMPs). Ils participent à la regulation du remodelage matriciel, mais présentent aussi des fonctions indépendantes de cette activité (facteur de croissance, facteur de survie). A travers ces multiples facettes, la protéine TIMP-1 est impliquée dans des processus phvsiologiques et pathologiques. Nos objectifs sont de caractériser et d'utiliser les propriétés biochimiques de TIMP-1 afin d'évaluer son potentiel thérapeutique. Nous avons établi un protocole d'expression en système mammifère puis un schéma de purification de la protéine rh-TIMP-1 (recombinant human TIMP-1) nous permettant d'obtenir 30 mg/l de TlMP-1 pure, dépassant largement les taux dejà décrits. Ceci nous a permis d'évaluer son efficacité en tant qu'agent thérapeutique dans un modèle murin d'arthrite au collagène (mimant la polyarthrite rhumatoïde). Des analvses cliniques et sérologiques suggèrent un effet bénéfique des injections de rh-TIMP-1 à dose élevée contrairement aux doses faibles. Des investigations supplémentaires sont nécessaires pour déterminer le réel effet de TIMP-1. En parallèle, nous avons étudié la propriété de transduction de TIMP-1 dans les cellules tumorales. Nous avons montré que les acides aminés C-terminaux portent cette propriété et désormais nous devons optimiser ce processus et tester le transport d'une protéine thérapeutique (p.53). Ce travail confirme l'intérêt biotechnologique de la proteine TIMP-1 en tant que navette et suggère l'intérêt thérapeutique potentiel qui nécessite des investigations additionnelles en particulier dans d'autres modèles pathologiques.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Théâtre de jeunesse et lecture de littérature au cycle 3 : donner voix et corps aux textes

International audienc

A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

International audienceThe dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models

Structural polymorphism of non-covalent peptide-based delivery systems: Highway to cellular uptake.

International audienceDuring the last two decades, delivery has become a major challenge for the development of new therapeutic molecules for the clinic. Although, several strategies either viral or non viral have been proposed to favor cellular uptake and targeting of therapeutics, only few of them have reach preclinical evaluation. Amongst them, cell-penetrating peptide (CPP) constitutes one of the most promising strategy and has applied for systemic in vivo delivery of a variety of therapeutic molecules. Two CPP-strategies have been described; using peptide carriers either covalently-linked to the cargo or forming non-covalent stable complexes with cargo. Peptide-based nanoparticle delivery system corresponds to small amphipathic peptides able to form stable nanoparticles with either proteins/peptides or nucleic acids and to enter the cell independently of the endosomal pathway. Three families of peptide-based nanoparticle systems; MPG, PEP and CADY have been successfully used for the delivery of various biologically active cargoes both ex vivo and in vivo in several animal models. This review will focus on the mechanism of the peptide-based nanoparticles; PEP, MPG and CADY in a structural and biophysical context. It will also highlight the major parameters associated to particle formation/stabilization and the impact of the carrier structural polymorphism in triggering cellular uptake

A non-covalent peptide-based strategy for siRNA delivery

International audienceThe major obstacle to clinical development of siRNAs (short interfering RNAs), like for most of the nucleic-acid-based strategies, is their poor cellular uptake and bioavailability. Although several viral and non-viral strategies have been proposed to improve siRNA delivery, their applications in vivo remain a major challenge. We have developed a new strategy, based on a short amphipathic peptide, MPG, that is able to form stable nanoparticles with siRNA. MPG-based particles enter the cell independently of the endosomal pathway and can efficiently deliver siRNA in a fully biologically active form into a variety of cell lines and in vivo. This short review will discuss the mechanism and the potency of the MPG strategy for siRNA delivery both in vitro and in vivo

Tentative de suicide par injection intraveineuse de mercure. À propos d’un cas

Objectif : Nous rapportons le cas d’une tentative de suicide par injection intraveineuse de mercure métal. Résultats : Les emboles de mercure sont retrouvés sur les radiographies et les scanners thoraciques et abdominaux au niveau des poumons, du cœur, des reins et du foie. Le mercure est dosé par SAA et ICP-MS dans le sang, les urines et les cheveux. Discussion : Lors du suivi deux années durant, malgré des concentrations de mercure élevées dans le sang et les urines, et la persistance de dépôts mercuriels dans les tissus, aucune anomalie biologique des fonctions hépatique ou rénale ni atteinte clinique pulmonaire et neurologique ne sont détectées