RESUMEN
Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is the formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g., blood stream. In this study, we compared protein corona of CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g., by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be considered while developing delivery systems for nucleic acids.
RESUMEN
Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.
Asunto(s)
Arginina/análisis , Péptidos de Penetración Celular/metabolismo , Colesterol/metabolismo , Inmunoconjugados/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/química , Ratones , Transporte de Proteínas , NucleolinaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Lipopéptidos/farmacología , Receptores Depuradores/metabolismo , Citoesqueleto de Actina/metabolismo , Señalización del Calcio , Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Células HeLa , Humanos , Lipopéptidos/química , Nanoestructuras/química , Ácidos Nucleicos/química , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.
Asunto(s)
Antiinflamatorios/administración & dosificación , Queratinocitos/efectos de los fármacos , Lipopéptidos/administración & dosificación , MicroARNs/administración & dosificación , Nanopartículas/administración & dosificación , Quinolinas/administración & dosificación , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/inmunología , Dermatitis por Contacto/tratamiento farmacológico , Dermatitis por Contacto/inmunología , Modelos Animales de Enfermedad , Humanos , Irritantes , Queratinocitos/metabolismo , Lipopéptidos/química , Lipopéptidos/uso terapéutico , Ratones Endogámicos C57BL , MicroARNs/química , MicroARNs/genética , MicroARNs/uso terapéutico , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/uso terapéutico , Nanopartículas/ultraestructura , Quinolinas/química , Quinolinas/uso terapéutico , Acetato de TetradecanoilforbolRESUMEN
Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. The elevated Ca(2+) concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca(2+) and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca(2+) if its level is not drastically elevated.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Exocitosis/efectos de los fármacos , Lisosomas/metabolismo , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Células HeLa , Humanos , Células JurkatRESUMEN
The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.
Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Membrana Dobles de Lípidos/química , Péptidos/química , Péptidos/farmacocinética , Membrana Celular/química , Membrana Celular/ultraestructura , Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Células HeLa , Humanos , Membrana Dobles de Lípidos/metabolismo , Péptidos/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismoRESUMEN
Despite the quickly widening application of cell-penetrating peptides (CPP) for the cellular delivery of various macromolecules, the cell entry mechanisms of these peptides have remained elusive so far. The basic features of the translocation of CPPs into cells have been mapped by fluorescence microscopy and activity-based assays revealing that endocytotic mechanisms are mainly responsible for the uptake at physiological temperature. However, the high concentration of CPP or the lowering of the incubation temperature below 10°C (re)activates a nonvesicular cell entry mode. The fluorescence microscopy can hardly provide detailed information about the interaction of CPP molecules with the extracellular structures, the induced changes in the morphology of the plasma membrane, etc. Therefore, application of electron microscopy could help to shed light on the nature of nonvesicular uptake mechanism. Transmission electron microscopy (TEM) has been a valuable tool for the morphological characterization of biological material at high resolution. It can provide useful information at the ultrastructural level about the interaction and arrangement of CPPs on the cell surface, the entrapment in cellular organelles and the translocation to the cytoplasm. In this chapter, we present a method for the tagging of CPPs covalently with a 1.4 nm gold cluster and provide a flat-embedding protocol for the mapping of Nanogold™-labeled CPPs in cultured cells by TEM. This method enables to retain the cell monolayers in their in situ orientation. The Nanogold™ tag is putatively not interfering with the uptake of CPPs and enables the production of specimens with excellent morphology and good contrast.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Endocitosis , Microscopía Electrónica de Transmisión/métodos , Péptidos de Penetración Celular/química , Oro/química , Células HeLa , Humanos , Nanopartículas del Metal/química , Plata/química , Coloración y EtiquetadoRESUMEN
Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.
Asunto(s)
Membrana Celular/metabolismo , Nanopartículas/química , Péptidos/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Células HeLa , Humanos , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Péptidos/química , Conformación Proteica , Temperatura , Distribución TisularRESUMEN
Delivery of large bioactive cargoes into cells with the help of cell-penetrating peptides (CPPs) is mostly based on endocytic processes. Here we map the cellular pathways used by transportan and transportan 10 (TP10) for protein transduction in HeLa cells. CPP-mediated cellular delivery is often suggested to be lipid-raft-dependent; therefore, we used flotillin-1, caveolin, Rab5, and PI3P as markers to elucidate the involvement of these particular endosomal pathways in the protein uptake process. Confocal laser scanning and electron microscopy reveal only a negligible overlap of avidin/neutravidin conveyed into cells by transportans with the raft marker flotillin-1 or early endosomal markers Rab5 and PI3P. However, about 20% of protein-CPP complexes colocalize with the caveolar/caveosomal marker caveolin, and down-regulation of caveolin-1 by siRNA treatment leads to the inhibition of the CPP-mediated protein uptake by 30-50%. On the contrary, the lack of flotillin-1 increases rather than decreases the CPP-mediated protein transport. The participation of the caveolin-1-dependent pathway in CPP-mediated protein delivery was also corroborated by using caveolin-1 knockout mouse embryonic fibroblasts.
Asunto(s)
Caveolas/metabolismo , Galanina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Venenos de Avispas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Caveolina 1/deficiencia , Caveolina 1/genética , Colesterol/metabolismo , Regulación hacia Abajo , Endosomas/metabolismo , Galanina/química , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/química , Venenos de Avispas/química , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis--perforin, granulysin, and granzymes--share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.