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

Caractérisation et optimisation des nanoparticules CADY/siRNA en vue d’une application in vivo

Cell penetrating peptides (CPPs) are short peptides that can enter many cell types and transduce into cells a wide range of molecular therapeutics (nucleic acid, proteins, peptides, small molecules, etc.). Our laboratory has developed the secondary amphipathic peptide CADY able to promote the transport of small interfering RNA (siRNA) independently of all endocytotic pathways. Indeed, siRNA therapeutic interest lies on its ability to inhibit specifically deregulated proteins in the context of pathology. The subject of my thesis focused on the characterization and optimization of CADY/siRNA complexes. During my work, we have been able to show that CADY adopts a helical structure while interacting with the siRNA leading to the formation of nanoparticles. The goal of my study was to optimize CADY sequence and control its formulation to consider the transferring from an in cellulo to an in vivo application of our vectorization system. First, we conducted a structure-activity study with six analogues by mutating CADY on tryptophane residues (PSF1, 2, 3 and PSW) and in the area initializing helical structure (PG9, PG16). A thorough analysis of these analogues has confirmed that the limitation of the amphipathic character and structural polymorphism is directly related to the reduction of internalization efficiency of our CPPs. Among the six analogues, only PG16/siRNA and PG9/siRNA nanoparticles show in cellulo results equal to those obtained with CADY/siRNA. Based on the fact, that CADY is the most suitable vector for the transfection of therapeutic molecules such as siRNA, we have established a standard formulation procedure to obtain reproducible and homogeneous CADY/siRNA complexes with an average size of 106 ± 31 nm and a polydispersity index of 0.357 ± 0.053. In addition, we have implemented an extrusion/lyophilization step to allow nanoparticle storage as powder, which can be re-suspended in an aqueous solution without losing their colloidal and transfection properties.In order to improve tissue specificity and bioavailability of CADY/siRNA nanoparticles for an in vivo application, we have grafted ether a targeting sequence (YIGSR-S) or a stealth motif (PEG) to the CADY sequence. These two entities of very different nature provoke only few changes in the physicochemical (e.g. average size) and biological (cell transfection) characteristics of the nanoparticles formed with a siRNA. These results are very encouraging for the development of the so-called 3rd nanoparticle generation which includes several kinds of molecules (targeting, polymer, contrast agent etc.).These outcomes mark the real progress in CADY formulation optimization, and encourage us to further exploit its potential for the in vivo transfer of siRNA.Les « cell penetrating peptides » (CPPs) sont des vecteurs peptidiques capables de délivrer diverses molécules (acides nucléiques, protéines, peptides, petites molécules, etc.) à l'intérieur des cellules de mammifères. Notre laboratoire a élaboré le vecteur amphipatique secondaire CADY capable de transporter, indépendamment de toute voie d'endocytose, des molécules thérapeutiques telles que les petits ARN interférents (siRNA). En effet, leur potentiel thérapeutique réside dans leur capacité à inhiber de manière spécifique l'expression de protéines dérégulées dans un cadre pathologique.Le sujet de ma thèse s'est centré sur la caractérisation et l'optimisation des complexes CADY/siRNA. Au cours de mes travaux, nous avons pu mettre en évidence que CADY adoptait une structure en hélice alpha en présence du siRNA ce qui conduit à la formation des nanoparticules. Notre étude a eu pour but d'optimiser la séquence de CADY et de contrôler sa formulation pour permettre le transfert de notre système de vectorisation d'une application in cellulo à une application in vivo. En premier lieu, nous avons mené une étude de relations structure-activité avec six peptides analogues de CADY, en réalisant des mutations sur les résidus tryptophanes (PSF1, PSF2, PSF3 et PSW) et sur la zone initiatrice de l'hélice alpha (PG9, PG16). L'analyse approfondie de ces analogues a permis de confirmer que la limitation du caractère amphipathique et du polymorphisme structural des vecteurs conduisaient à une réduction de l'efficacité d'internalisation. Parmi les 6 analogues, seules les nanoparticules à base de PG9 et PG16 présentent des résultats in cellulo comparables à ceux obtenus avec les nanoparticules CADY/siRNA. A ce jour, la séquence primaire de CADY étant la plus adaptée pour la transfection de siRNA, nous avons établi une procédure de formulation standardisée permettant un autoassemblage CADY/siRNA reproductible et homogène, dont la taille moyenne est de 106 ± 31 nm et l'indice de polydispersité de 0,357 ± 0,053. De plus, nous avons mis en place une procédure d'extrusion/lyophilisation afin de stocker les nanoparticules sous forme de poudre. Celle-ci peut être resuspendue en milieu aqueux sans modifications des propriétés colloïdales des nanoparticules ni de leur capacité de transfection.Dans le but d'améliorer la spécificité tissulaire et la biodisponibilité des nanoparticules CADY/siRNA pour une application in vivo, nous avons greffé des motifs de ciblage (YIGSR-S) et de furtivité (PEG) sur la séquence de CADY. L'ajout de ces deux entités, de natures très différentes, ne modifie que faiblement les caractéristiques physico-chimiques (ex. taille moyenne des complexes) et biologiques (transfection cellulaire) des nanoparticules. Ces résultats sont très encourageants pour le développement de nanoparticules dites de 3ème génération, sur lesquelles on peut greffer plusieurs sortes de molécules d'intérêts (ciblage, polymère, agent de contraste etc.).L'ensemble des résultats obtenus au cours de ma thèse marque un réel progrès dans l'optimisation de la formulation du vecteur CADY, et nous incitent à exploiter davantage son potentiel pour le transfert de siRNA in vivo

Characterization and optimization of CADY/siRNA nanoparticles for an in vivo application

Les « cell penetrating peptides » (CPPs) sont des vecteurs peptidiques capables de délivrer diverses molécules (acides nucléiques, protéines, peptides, petites molécules, etc.) à l'intérieur des cellules de mammifères. Notre laboratoire a élaboré le vecteur amphipatique secondaire CADY capable de transporter, indépendamment de toute voie d'endocytose, des molécules thérapeutiques telles que les petits ARN interférents (siRNA). En effet, leur potentiel thérapeutique réside dans leur capacité à inhiber de manière spécifique l'expression de protéines dérégulées dans un cadre pathologique.Le sujet de ma thèse s'est centré sur la caractérisation et l'optimisation des complexes CADY/siRNA. Au cours de mes travaux, nous avons pu mettre en évidence que CADY adoptait une structure en hélice alpha en présence du siRNA ce qui conduit à la formation des nanoparticules. Notre étude a eu pour but d'optimiser la séquence de CADY et de contrôler sa formulation pour permettre le transfert de notre système de vectorisation d'une application in cellulo à une application in vivo. En premier lieu, nous avons mené une étude de relations structure-activité avec six peptides analogues de CADY, en réalisant des mutations sur les résidus tryptophanes (PSF1, PSF2, PSF3 et PSW) et sur la zone initiatrice de l'hélice alpha (PG9, PG16). L'analyse approfondie de ces analogues a permis de confirmer que la limitation du caractère amphipathique et du polymorphisme structural des vecteurs conduisaient à une réduction de l'efficacité d'internalisation. Parmi les 6 analogues, seules les nanoparticules à base de PG9 et PG16 présentent des résultats in cellulo comparables à ceux obtenus avec les nanoparticules CADY/siRNA. A ce jour, la séquence primaire de CADY étant la plus adaptée pour la transfection de siRNA, nous avons établi une procédure de formulation standardisée permettant un autoassemblage CADY/siRNA reproductible et homogène, dont la taille moyenne est de 106 ± 31 nm et l'indice de polydispersité de 0,357 ± 0,053. De plus, nous avons mis en place une procédure d'extrusion/lyophilisation afin de stocker les nanoparticules sous forme de poudre. Celle-ci peut être resuspendue en milieu aqueux sans modifications des propriétés colloïdales des nanoparticules ni de leur capacité de transfection.Dans le but d'améliorer la spécificité tissulaire et la biodisponibilité des nanoparticules CADY/siRNA pour une application in vivo, nous avons greffé des motifs de ciblage (YIGSR-S) et de furtivité (PEG) sur la séquence de CADY. L'ajout de ces deux entités, de natures très différentes, ne modifie que faiblement les caractéristiques physico-chimiques (ex. taille moyenne des complexes) et biologiques (transfection cellulaire) des nanoparticules. Ces résultats sont très encourageants pour le développement de nanoparticules dites de 3ème génération, sur lesquelles on peut greffer plusieurs sortes de molécules d'intérêts (ciblage, polymère, agent de contraste etc.).L'ensemble des résultats obtenus au cours de ma thèse marque un réel progrès dans l'optimisation de la formulation du vecteur CADY, et nous incitent à exploiter davantage son potentiel pour le transfert de siRNA in vivo.Cell penetrating peptides (CPPs) are short peptides that can enter many cell types and transduce into cells a wide range of molecular therapeutics (nucleic acid, proteins, peptides, small molecules, etc.). Our laboratory has developed the secondary amphipathic peptide CADY able to promote the transport of small interfering RNA (siRNA) independently of all endocytotic pathways. Indeed, siRNA therapeutic interest lies on its ability to inhibit specifically deregulated proteins in the context of pathology. The subject of my thesis focused on the characterization and optimization of CADY/siRNA complexes. During my work, we have been able to show that CADY adopts a helical structure while interacting with the siRNA leading to the formation of nanoparticles. The goal of my study was to optimize CADY sequence and control its formulation to consider the transferring from an in cellulo to an in vivo application of our vectorization system. First, we conducted a structure-activity study with six analogues by mutating CADY on tryptophane residues (PSF1, 2, 3 and PSW) and in the area initializing helical structure (PG9, PG16). A thorough analysis of these analogues has confirmed that the limitation of the amphipathic character and structural polymorphism is directly related to the reduction of internalization efficiency of our CPPs. Among the six analogues, only PG16/siRNA and PG9/siRNA nanoparticles show in cellulo results equal to those obtained with CADY/siRNA. Based on the fact, that CADY is the most suitable vector for the transfection of therapeutic molecules such as siRNA, we have established a standard formulation procedure to obtain reproducible and homogeneous CADY/siRNA complexes with an average size of 106 ± 31 nm and a polydispersity index of 0.357 ± 0.053. In addition, we have implemented an extrusion/lyophilization step to allow nanoparticle storage as powder, which can be re-suspended in an aqueous solution without losing their colloidal and transfection properties.In order to improve tissue specificity and bioavailability of CADY/siRNA nanoparticles for an in vivo application, we have grafted ether a targeting sequence (YIGSR-S) or a stealth motif (PEG) to the CADY sequence. These two entities of very different nature provoke only few changes in the physicochemical (e.g. average size) and biological (cell transfection) characteristics of the nanoparticles formed with a siRNA. These results are very encouraging for the development of the so-called 3rd nanoparticle generation which includes several kinds of molecules (targeting, polymer, contrast agent etc.).These outcomes mark the real progress in CADY formulation optimization, and encourage us to further exploit its potential for the in vivo transfer of siRNA

Interactions of amphipathic CPPs with model membranes.

International audienceDue to the poor permeability of the plasma membrane, several strategies are designed to enhance the transfer of therapeutics into cells. Over the last 20 years, small peptides called Cell-Penetrating Peptides (CPPs) have been widely developed to improve the cellular delivery of biomolecules. These small peptides derive from protein transduction domains, chimerical constructs, or model sequences. Several CPPs are primary or secondary amphipathic peptides, depending on whether the distribution of their hydrophobic and hydrophilic domains occurs from their amino-acid sequence or through α-helical folding. Most of the CPPs are able to deliver different therapeutics such as nucleic acids or proteins in vitro and in vivo. Although their mechanisms of internalization are varied and controversial, the understanding of the intrinsic features of CPPs is essential for future developments. This chapter describes several protocols for the investigation of biophysical properties of amphipathic CPPs. Surface physics approaches are specifically applied to characterize the interactions of amphipathic peptides with model membranes. Circular dichroism and infra-red spectroscopy allow the identification of their structural state. These methods are exemplified by the analyses of the main biophysical features of the cell-penetrating peptides MPG, Pep-1, and CADY

Design of multifunctional peptide-based nanoparticles for specific cell-delivery of siRNAs

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Secondary structure of cell-penetrating peptides controls membrane interaction and insertion.

International audienceThe clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature

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

In Vivo Follow-Up of Gene Inhibition in Solid Tumors Using Peptide-Based Nanoparticles for siRNA Delivery

International audienceSmall interfering RNA (siRNA) exhibits a high degree of specificity for targeting selected genes. They are efficient on cells in vitro, but in vivo siRNA therapy remains a challenge for solid tumor treatment as siRNAs display difficulty reaching their intracellular target. The present study was designed to show the in vivo efficiency of a new peptide (WRAP5), able to form peptide-based nanoparticles (PBN) that can deliver siRNA to cancer cells in solid tumors. WRAP5:siRNA nanoparticles targeting firefly luciferase (Fluc) were formulated and assayed on Fluc-expressing U87 glioblastoma cells. The mode of action of WRAP5:siRNA by RNA interference was first confirmed in vitro and then investigated in vivo using a combination of bioluminescent reporter genes. Finally, histological analyses were performed to elucidate the cell specificity of this PBN in the context of brain tumors. In vitro and in vivo results showed efficient knock-down of Fluc expression with no toxicity. WRAP5:siFluc remained in the tumor for at least 10 days in vivo. Messenger RNA (mRNA) analyses indicated a specific decrease in Fluc mRNA without affecting tumor growth. Histological studies identified PBN accumulation in the cytoplasm of tumor cells but also in glial and neuronal cells. Through in vivo molecular imaging, our findings established the proof of concept for specific gene silencing in solid tumors. The evidence generated could be translated into therapy for any specific gene in different types of tumors without cell type specificity but with high molecular specificit

Optimization of peptide-plasmid DNA vectors formulation for gene delivery in cancer therapy exploring design of experiments

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