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Alzheimer's disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-ß (Aß) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aß. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aß interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aß aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aß-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.
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Doença de Alzheimer , Peptídeos Penetradores de Células , Doenças Neurodegenerativas , Animais , Humanos , Peptídeos Penetradores de Células/uso terapêutico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sinais Direcionadores de Proteínas , Proteínas tau/metabolismo , Mamíferos/metabolismoRESUMO
Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a "kinked" structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.
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mRNA-based therapeutics are presently one of the nucleic acid-based therapeutics with a high potential for extraordinary success as preventive vaccines. Current applications with mRNA therapeutics rely on lipid nanoparticle (LNP) mediated delivery of nucleic acids. In order to achieve the transition from preventive to therapeutic vaccines, there is a challenge of delivering the mRNA into non-hepatic tissues, especially into lymphoid tissues such as the spleen and lymph nodes. In this work, we characterize new cell-penetrating peptides NF424 and NF436 that exhibit preferential delivery of mRNA into the spleen after a single i.v. injection, without the use of any active targeting mechanisms. We show that between the spleen, liver, and the lungs, >95% of mRNA expression arises in the spleen tissue and the majority of expression occurs in the dendritic cells. The cell-penetrating peptides NF424 and NF436 represent promising candidates for cancer immunotherapeutic applications with tumor antigens.
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Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.
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We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10324-7.
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The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but the distribution of the nanoparticles in organisms is mostly determined by their physical properties. Therefore, generation of stable particles with strictly defined characteristics is highly essential. Here we describe a formulation protocol of stable and homogenous CPP/pDNA nanoparticles for in vivo applications.
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Nanopartículas , Peptídeos Penetradores de Células , PlasmídeosRESUMO
Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.
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Peptides can be used as research tools and for diagnostic or therapeutic applications. Peptides, alongside small molecules and antibodies, are used and are gaining further interest as protein-protein interaction (PPI) modulators. Peptides have high target specificity and high affinity, but, unlike small molecule modulators, they are not able to cross the cell membranes to reach their intracellular targets. To overcome this limitation, the special property of the cell-penetrating peptides (CPPs) could benefit their cause. CPPs are a class of peptides that can enter the cells and with them also deliver the attached cargoes. Today, with the advancement of in silico prediction tools and the availability of protein databases, designing new and multifunctional peptides that are able to reach intracellular targets and inhibit certain cellular processes in a very specific manner is reachable. Although there are several efficient CPP sequences already known, the discovery of new CPPs is crucial for the development of efficient delivery methods for both biotechnological and therapeutic applications. In this work, we chose 10 human nuclear proteins from which we predicted new potential CPP sequences by using three different CPP predictors: cell-penetrating peptide prediction tool, CellPPD, and SkipCPP-Pred. From each protein, one predicted CPP sequence was synthesized and its internalization into cells was assessed. Out of the tested sequences, three peptides displayed features characteristic to CPPs. These peptides and also the predicted peptide sequences could be used to design and modify new CPPs. In this work, we show that we can use protein sequences as input for generating new peptides with cell internalization properties. Three new CPPs, AHRR8-24, CASC3251-264, and AKIP127-37, can be further used for the delivery of other cargoes or designed into multifunctional peptides with capability of internalizing cells.
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MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1ß, IL-33 and TNF-α. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.
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Peptídeos Penetradores de Células , MicroRNAs , Animais , Células Dendríticas , Inflamação , Queratinócitos , Camundongos , MicroRNAs/genéticaRESUMO
Cell-penetrating peptides (CPPs) are relatively short peptides that can enter to the cell interior and facilitate intracellular delivery of associated cargo molecules. NickFects is a novel family of CPPs, designed to deliver various types of bio-active cargos using non-covalent nanoparticle formation approach. This chapter describes in details the manual synthesis of cell-penetrating peptides using Fmoc-solid phase peptide synthesis (SPPS).
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Aminoácidos/química , Peptídeos Penetradores de Células/síntese química , Técnicas de Síntese em Fase Sólida , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/isolamento & purificação , Técnicas de Química Sintética , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/química , Humanos , Nanopartículas/química , Acoplamento OxidativoRESUMO
Cationic peptides designed for cellular delivery of nucleic acid molecules form noncovalent nanocomplexes with negatively charged oligonucleotides (ON). The electrostatically associated complexes are further compacted by hydrophobic interactions yielding nanoparticles (NP) of homogeneous shape and size that are efficiently taken up by cells. The shape and size of NP often correlate with the biological activity of delivered ON inside cells; and the stability and accessibility of NP in biological fluids govern its circulation in organism and the cellular uptake. Therefore, here we provide protocols for characterizing the shape and size and surface charge of peptide/ON NP by negative staining transmission electron microscopy (TEM) and dynamic light scattering (DLS) respectively, and analysis of NP stability against proteolytic degradation.
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Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Oligonucleotídeos/química , Peptídeos/química , Difusão Dinâmica da Luz , Endopeptidases/química , Humanos , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Nanopartículas/ultraestrutura , ProteóliseRESUMO
Extracellular synthetic nucleic acids, such as siRNAs, are unable to reach their intended targets efficiently. Therefore, delivery methods such as cell-penetrating peptides (CPP), which increase their transport, could enhance the potency of siRNA as therapeutic agents. The CPP NickFect55 (NF55) is an efficient peptide-based delivery vector, which has been previously used to deliver plasmid DNA into cells in vivo. To achieve higher intracellular delivery and bioactivity from the delivered cargo, we designed a series of histidine-containing peptides by optimizing pH-sensitivity, net charge, hydrophobicity, and charge distribution in the sequence of the CPP NF55. In the current work, we have applied a strategy where we have replaced amino acids in the C-terminus of the peptide in order to distribute hydrophobic and hydrophilic amino acids into distinct regions along the alpha-helical projection, including histidine amino acids into the sequence at the N-terminus, and optimizing the N-terminal fatty acid modification to suit the specific peptide sequence. We tested the CPPs based on the transfection efficacy, CPP/siRNA complex stability, and the properties of the CPPs, such as hemolytic activity, buffering capability and cell toxicity. As a result, we have introduced a new peptide with a completely redesigned N-terminus that displays adaptive response to its physical environment. This peptide - NickFect70 (NF70) - efficiently condenses siRNA, protects the cargo against degradation and effectively mediates target gene knockdown both in mammalian cell culture and in vivo, in a mouse model.
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Peptídeos Penetradores de Células/metabolismo , Ácidos Graxos/metabolismo , Histidina/metabolismo , Animais , Peptídeos Penetradores de Células/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/genéticaRESUMO
Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.
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Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.
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Peptídeos Penetradores de Células/genética , Ácidos Graxos/química , Lipopeptídeos/genética , Transfecção/métodos , Animais , Células CHO , Peptídeos Penetradores de Células/química , Cricetinae , Cricetulus , Difusão Dinâmica da Luz , Feminino , Corantes Fluorescentes/química , Lipopeptídeos/química , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/químicaRESUMO
Cell-penetrating peptides (CPPs) are delivery vectors widely used to aid the transport of biologically active cargoes to intracellular targets. These cargoes include small interfering RNAs (siRNA) that are not naturally internalized by cells. Elucidating the complexities behind the formation of CPP and cargo complexes is crucial for understanding the processes related to their delivery. In this study, we used modified analogs of the CPP transportan10 and investigated the binding properties of these CPPs to siRNA, the formation parameters of the CPP/siRNA complexes, and their stabiliy to enzymatic degradation. We conclude that the pH dependent change of the net charge of the CPP may very well be the key factor leading to the high delivery efficiency and the optimal binding strength between CPPs to siRNAs, while the hydrophobicity, secondary structure of the CPP, and the positions of the positive charges are responsible for the stability of the CPP/siRNA particles. Also, CPPs with distinct hydrophobic and hydrophilic regions may assemble into nanoparticles that could be described as core-shell formulations.
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Scavenger receptors (SRs) are a large family of multifunctional receptors that are involved in a range of physiologic and pathologic processes. The ability of class A scavenger receptors (SR-As) to bind anionic ligands facilitates the internalization of negatively charged cell-penetrating peptide (CPP)-nucleic acid nanocomplexes and thus makes them attractive targets for delivery of various nucleic acids. Recently, we demonstrated that SR-A3 and SR-A5 are recruited from intracellular membranes to the plasma membrane after incubation with PepFect 14-splice-switching oligonucleotide complexes. Here, we examined the mechanisms responsible for translocation of SR-As to the cell surface. We demonstrate that, in addition to nanocomplexes, some amphipathic CPPs are able to induce externalization of SR-A3 and SR-A5, and this process requires the presence of calcium ions. Furthermore, translocation of SR-A3 and SR-A5 requires activity of phosphatidylinositol-3-kinase, intact actin cytoskeleton, and the presence of serum proteins in culture medium.-Juks, C., Lorents, A., Arukuusk, P., Langel, Ü., Pooga, M. Cell-penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids.
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Membrana Celular/metabolismo , Peptídeos Penetradores de Células/farmacologia , Lipopeptídeos/farmacologia , Receptores Depuradores/metabolismo , Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Células HeLa , Humanos , Lipopeptídeos/química , Nanoestruturas/química , Ácidos Nucleicos/química , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.
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Trifosfato de Adenosina/química , Proteínas Quinases Dependentes de AMP Cíclico/química , AMP Cíclico/química , Peptídeos/química , Inibidores de Proteínas Quinases/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Corantes Fluorescentes/química , Humanos , Cinética , Ligantes , Peptídeos/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Coloração e Rotulagem/métodos , TermodinâmicaRESUMO
As the field of gene therapy progresses, an increasingly urgent need has arisen for efficient and non-toxic vectors for the in vivo delivery of nucleic acids. Cell-penetrating peptides (CPP) are very efficient transfection reagents in vitro, however, their application in vivo needs improvement. To enhance in vivo transfection we designed various CPPs based on previous knowledge of internalization studies and physiochemical properties of NickFect (NF) nanoparticles. We show that increment of the helicity of these Transportan10 analogues improves the transfection efficiency. We rationally design by modifying the net charge and the helicity of the CPP a novel amphipathic α-helical peptide NF55 for in vivo application. NF55 condenses DNA into stable nanoparticles that are resistant to protease degradation, promotes endosomal escape, and transfects the majority of cells in a large cell population. We demonstrate that NF55 mediates DNA delivery in vivo with gene induction efficiency that is comparable to commercial transfection reagents. In addition to gene induction in healthy mice, NF55/DNA nanoparticles showed promising tumor transfection in various mouse tumor models, including an intracranial glioblastoma model. The efficiency of NF55 to convey DNA specifically into tumor tissue increased even further after coupling a PEG2000 to the peptide via a disulphide-bond. Furthermore, a solid formulation of NF55/DNA displayed an excellent stability profile without additives or special storage conditions. Together, its high transfection efficacy and stability profile make NF55 an excellent vector for the delivery of DNA in vivo.
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Peptídeos Penetradores de Células/química , DNA/administração & dosagem , Portadores de Fármacos/química , Técnicas de Transferência de Genes , Nanopartículas/química , Sequência de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Plasmídeos , Conformação Proteica , TransfecçãoRESUMO
Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.
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Peptídeos Penetradores de Células/metabolismo , Glicosaminoglicanos/fisiologia , Vesículas Transportadoras/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Galanina/metabolismo , Heparina Liase/farmacologia , Humanos , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Receptores Adrenérgicos beta 1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Transportadoras/química , Venenos de Vespas/metabolismo , Aglutininas do Germe de Trigo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems.