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1.
Int J Mol Sci ; 24(8)2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37108337

RESUMO

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is mainly caused by an oncogenic mutation in the KIT receptor tyrosine kinase. Targeting KIT using tyrosine kinase inhibitors, such as imatinib and sunitinib, provides substantial benefit; however, in most patients, the disease will eventually progress due to KIT secondary mutations leading to treatment failure. Understanding how GIST cells initially adapt to KIT inhibition should guide the selection of appropriate therapies to overcome the emergence of resistance. Several mechanisms have been broadly implicated in the resistance to imatinib anti-tumoral effects, including the reactivation of MAPK signaling upon KIT/PDGFRA targeted inhibition. This study provides evidence that LImb eXpression 1 (LIX1), a protein we identified as a regulator of the Hippo transducers YAP1 and TAZ, is upregulated upon imatinib or sunitinib treatment. LIX1 silencing in GIST-T1 cells impaired imatinib-induced MAPK signaling reactivation and enhanced imatinib anti-tumor effect. Our findings identified LIX1 as a key regulator of the early adaptative response of GIST cells to targeted therapies.


Assuntos
Antineoplásicos , Tumores do Estroma Gastrointestinal , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Relacionadas à Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Sistema de Sinalização das MAP Quinases
2.
J Nanobiotechnology ; 19(1): 236, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380479

RESUMO

Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.


Assuntos
Bandagens Compressivas , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , Transfecção , Sequência de Aminoácidos , Linhagem Celular Tumoral , Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos , Inativação Gênica/efeitos dos fármacos , Glioblastoma , Humanos , Indicadores e Reagentes/química , RNA Interferente Pequeno/farmacologia , Relação Estrutura-Atividade
3.
Bioconjug Chem ; 30(3): 592-603, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30586303

RESUMO

Delivery of small interfering RNA (siRNA) as a therapeutic tool is limited due to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule, and its instability in serum. Several siRNA delivery systems have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells, especially by forming nanoparticles. In this study, we have developed a new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells. The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as characterized by biophysical methods. Furthermore, they have several benefits as oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA delivery in several cell types, and the high gene silencing activity, even in the presence of serum. In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA delivery through nanoparticle formation. Our results indicate that the WRAP family has significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.


Assuntos
Peptídeos Penetradores de Células/química , Nanopartículas/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , RNA Interferente Pequeno/genética , Transfecção
4.
J Nanobiotechnology ; 15(1): 34, 2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28454579

RESUMO

BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.


Assuntos
Peptídeos Penetradores de Células/química , Nanopartículas/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/metabolismo , Genes Reporter , Humanos , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
5.
Biochim Biophys Acta ; 1828(2): 499-509, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23000699

RESUMO

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY-siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14±1 CADYs is required to saturate a siRNA as compared to the 12±2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY-CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY-siRNA complexes.


Assuntos
Peptídeos Penetradores de Células/química , Peptídeos/química , RNA Interferente Pequeno/metabolismo , Motivos de Aminoácidos , Arginina/química , Vetores Genéticos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Eletricidade Estática , Termodinâmica , Fatores de Tempo
6.
Methods Mol Biol ; 2383: 475-490, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34766308

RESUMO

Cell-penetrating peptide (CPP)-based approaches are excellent method for delivering cell-impermeable compounds/therapeutics such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, and drugs, as covalently or noncovalently conjugated cargo into cells. Nowadays, it is generally accepted that cellular internalization of these CPP-cargoes or CPP-nanoparticles occur via endocytosis-dependent mechanisms or by direct cell translocation.Here, we describe a subset of biophysical and biological methods which can be used to dissect the internalization mechanism of CPPs. Presented protocols and results were shown for the recently developed siRNA-loaded WRAP-based nanoparticles. The rapid and efficient cell delivery of WRAP encapsulated siRNA could be attributed to the main direct cellular translocation of the nanoparticles even if, to some extent, endocytosis-dependent internalization occurred.Deciphering the internalization mechanism is still an important requirement to understand and to optimize the action mode of CPPs or CPP-based nanoparticles as transfection reagents.


Assuntos
Nanopartículas , Peptídeos Penetradores de Células , Endocitose , RNA Interferente Pequeno/genética , Transfecção
7.
Biochim Biophys Acta ; 1798(6): 1119-28, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20214875

RESUMO

The 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.


Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/química , Sistemas de Liberação de Medicamentos , Peptídeos/química , Fosfolipídeos/química , Animais , Membrana Celular/metabolismo , Humanos , Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Relação Estrutura-Atividade
8.
Biochim Biophys Acta ; 1798(12): 2304-14, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20541523

RESUMO

During 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.


Assuntos
Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Humanos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
9.
Biomedicines ; 9(5)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065544

RESUMO

Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV.

10.
Pharmaceutics ; 13(5)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069377

RESUMO

Small 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 specificity.

11.
Biochemistry ; 49(16): 3393-402, 2010 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-20302329

RESUMO

Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.


Assuntos
Peptídeos/química , RNA Interferente Pequeno/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Dicroísmo Circular , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Distribuição Normal , Oligorribonucleotídeos/química , Peptídeos/síntese química , Peptídeos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína/genética , RNA Interferente Pequeno/metabolismo
12.
J Vis Exp ; (166)2020 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-33393518

RESUMO

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.


Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Arginina/química , Fluorescência , Bicamadas Lipídicas/química , Nanopartículas/química , Peptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Lipossomas Unilamelares/química
13.
Biochim Biophys Acta Biomembr ; 1862(6): 183252, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32135145

RESUMO

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM). In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells. In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.


Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Endocitose , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular , Peptídeos Penetradores de Células/metabolismo , Inativação Gênica , Humanos
14.
Biochim Biophys Acta ; 1778(5): 1197-205, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18316038

RESUMO

Despite numerous investigations, the important structural features of Cell Penetrating Peptides (CPPs) remain unclear as demonstrated by the difficulties encountered in designing new molecules. In this study, we focused our interest on Penetratin and Transportan and several of their variants. Penetratin W48F and Penetratin W48F/W56F exhibit a reduced and a complete lack of cellular uptake, respectively; TP07 and TP10 present a similar cellular uptake as Transportan and TP08, TP13 and TP15 display no or weak internalization capacity. We applied the algorithmic method named PepLook to analyze the peptide polymorphism. The study reveals common conformational characteristics for the CPPs and their permeable variants: they all are polymorphic. Negative, non permeable, mutants share the opposite feature since they are monomorphic. Finally, we support the hypothesis that structural polymorphism may be crucial since it provides peptides with the possibility of adapting their conformation to medium hydrophobicity and or to partner diversity.


Assuntos
Proteínas de Transporte/química , Galanina/química , Polimorfismo Genético , Proteínas Recombinantes de Fusão/química , Venenos de Vespas/química , Sequência de Aminoácidos , Peptídeos Penetradores de Células , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
15.
Biol Cell ; 100(4): 201-17, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18341479

RESUMO

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.


Assuntos
Portadores de Fármacos/química , Peptídeos/farmacocinética , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Sistemas de Liberação de Medicamentos , Humanos , Dados de Sequência Molecular , Nanopartículas , Oligopeptídeos/química , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/uso terapêutico , Peptídeos/química , Peptídeos/uso terapêutico , Relação Estrutura-Atividade
16.
Biochim Biophys Acta Biomembr ; 1861(9): 1533-1545, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283917

RESUMO

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods. We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates. Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification.


Assuntos
Peptídeos Penetradores de Células/química , Espectrometria de Fluorescência/métodos , Triptofano/química , Animais , Transporte Biológico , Células CHO , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/metabolismo , Cricetulus , Endocitose , Fluorescência , Transporte Proteico
17.
Adv Drug Deliv Rev ; 60(4-5): 537-47, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18037526

RESUMO

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs), which have been successfully applied for in vivo delivery of biomolecules and constitute very promising tools. Distinct families of CPPs have been described; some require chemical linkage between the drug and the carrier for cellular drug internalization while others like Pep-and MPG-families, form stable complexes with drugs depending on their chemical nature. Pep and MPG are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG and Pep based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes in a fully biologically active form into a large variety of cell lines as well as in animal models. This review will focus on the mechanisms of non-covalent MPG and Pep-1 strategies and their applications in cultured cells and animal models.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Ácidos Nucleicos/administração & dosagem , Peptídeos/administração & dosagem , Proteínas/química , Animais , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos/tendências , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacocinética , Peptídeos/química , Peptídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética
18.
Biochim Biophys Acta ; 1758(3): 328-35, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16277976

RESUMO

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.


Assuntos
Cisteamina/análogos & derivados , Proteínas de Ligação a DNA/química , Portadores de Fármacos/química , Membranas Artificiais , Modelos Biológicos , Peptídeos/química , Sequência de Aminoácidos , Cisteamina/química , Dados de Sequência Molecular , Conformação Proteica , Transporte Proteico
19.
Biochim Biophys Acta ; 1758(11): 1846-51, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17011511

RESUMO

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs) CH3-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH, CH3-CO-GALFLAFLAAALS LMGLWSQPKKKRKV-NH-CH2-CH2-SH, and CH3-CO-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH called Pbeta, Palpha and Pep-1, respectively, to promote pore formation is examined both in Xenopus oocytes and artificial planar lipid bilayers. A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. This work shows that the cell-penetrating peptides Pbeta and Pep-1 are able to induce formation of transmembrane pores in artificial bilayers and that these pores are most likely at the basis of their ability to facilitate intracellular delivery of therapeutics. In addition, their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process.


Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fenômenos Fisiológicos Celulares , Canais Iônicos/metabolismo , Peptídeos/metabolismo , Animais , Membrana Celular/química , Permeabilidade da Membrana Celular/fisiologia , Núcleo Celular/química , Canais Iônicos/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Químicos , Oócitos/metabolismo , Peptídeos/química , Xenopus
20.
Biochim Biophys Acta ; 1758(3): 384-93, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16545342

RESUMO

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.


Assuntos
Cisteamina/análogos & derivados , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Ácidos Nucleicos Peptídicos/administração & dosagem , Peptídeos/administração & dosagem , Proteínas/administração & dosagem , Animais , Células Cultivadas , Cisteamina/administração & dosagem , Cisteamina/química , Cisteamina/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Humanos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Transporte Proteico , Proteínas/química , Proteínas/metabolismo
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