RESUMEN
Cellular and nuclear delivery of biomolecules is limited by low membrane permeability. Cell-penetrating peptides (CPPs) can be covalently linked to cargos to improve cellular internalization. Our work indicates that arginine-rich CPPs are also able to interact with a variety of cargos, including DNA, RNA, proteins and nanomaterials, in a noncovalent manner and subsequently effect their delivery into cells. The advantages of noncovalent attachment in CPP-mediated transduction are multiple: ease of use, ease of production, and versatility with respect to both cargo composition and functional delivery (i.e., the cargo is not chemically modified). We have extended this approach to achieve simultaneous transduction of covalently and noncovalently associated complexes, opening a new method for delivering multiple types of cargos, including proteins, fluorescent nanomaterials, nucleic acid and others. These novel variations of CPP-mediated transport should be of broad utility in the transport of genes, small interfering RNAs, proteins and nanoparticles in biomedical research and therapeutic intervention.
Asunto(s)
Péptidos de Penetración Celular/farmacocinética , Sistemas de Liberación de Medicamentos , Animales , Transporte Biológico , Péptidos de Penetración Celular/química , ADN/administración & dosificación , ADN/farmacocinética , Humanos , Nanoestructuras/administración & dosificación , Proteínas/administración & dosificación , Proteínas/farmacocinética , ARN/administración & dosificación , ARN/farmacocinéticaRESUMEN
DNA can be used to nanofabricate three-dimensional (3D) polyhedra. A variety of applications of 3D DNA assemblies have been proposed. Drug encasulation and intracellular delivery using DNA nanoparticles, however, have remained a challenge. Here, we create a distinct five-point-star motif and aptamer-conjugated six-point-star motif using well-used primer sequences to intermolecularly construct DNA icosahedra as a nanocarrier for doxorubicin. Aptamer-conjugated doxorubicin-intercalated DNA icosahedra (Doxo@Apt-DNA-icosa) show an efficient and specific internalization for killing epithelial cancer cells.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , ADN/química , ADN/metabolismo , Doxorrubicina/farmacología , Portadores de Fármacos/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Animales , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , ADN/genética , Doxorrubicina/uso terapéutico , Portadores de Fármacos/metabolismo , Humanos , Nanotecnología , Neoplasias/patologíaRESUMEN
Ultrasmall superparamagnetic iron oxide (USPIO) particles are very useful for cellular magnetic resonance imaging (MRI), which plays a key role in developing successful stem cell therapies. However, their low intracellular labeling efficiency, and biosafety concerns associated with their use, have limited their potential usage. In this study we develop a novel system composed of RBC-derived vesicles (RDVs) for efficient delivery of USPIO particles into human bone marrow mesenchymal stem cells (MSCs) for cellular MRI in vitro and in vivo. RDVs are highly biosafe to their autologous MSCs as manifested by cell viability, differentiation, and gene microarray assays. The data demonstrate the potential of RDVs as intracellular delivery vehicles for biomedical applications.
Asunto(s)
Compuestos Férricos/administración & dosificación , Imagen por Resonancia Magnética/métodos , Células Madre/citología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Eritrocitos/citología , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Células Madre/metabolismoRESUMEN
DNA molecular weight markers are routinely used in agarose gel electrophoresis. Here we report a method called PCR-synthesized marker (PSM) to generate DNA molecular rulers by PCR in the laboratory. This strategy can also be used to produce 100 bp RNA molecular weight markers by run-off transcription.
Asunto(s)
ADN/química , ADN/genética , Peso MolecularRESUMEN
The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.
Asunto(s)
Vectores Genéticos , Péptidos/metabolismo , Plantas/genética , Protoplastos/metabolismo , Transfección/métodos , Arginina/química , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos/química , Péptidos/química , Plantas Modificadas Genéticamente , Plásmidos/metabolismo , Factores de TiempoRESUMEN
* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos/metabolismo , Pinocitosis , Plantas/metabolismo , Transporte de Proteínas , Proteínas Portadoras/química , Línea Celular Tumoral , Técnicas Citológicas/métodos , Humanos , Cebollas/metabolismo , Péptidos/química , Plásmidos , Estructura Terciaria de Proteína , Proteínas/metabolismo , Zea mays/metabolismoRESUMEN
Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or beta-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.
Asunto(s)
Arginina/metabolismo , Células/citología , Células/metabolismo , Péptidos/química , Péptidos/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Cricetinae , Humanos , Hígado/citología , Hígado/metabolismo , Ratones , Cebollas/citología , Cebollas/metabolismo , Péptidos/genética , Péptidos/toxicidad , Plásmidos/genética , Transporte de Proteínas , Piel/citología , Piel/metabolismoRESUMEN
Protein molecular weight markers are widely used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Here, we describe novel protein molecular weight markers in which a prestaining procedure is no longer needed. Green fluorescent protein (GFP) is stable and resistant to denaturing agents/conditions. Various histidine-tagged GFP fusion proteins were overexpressed in Escherichia coli and purified by metal affinity chromatography. The minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot under visible light was 62.5 and 125 ng, respectively. Under ultraviolet (UV) ray, the minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot was half of those amounts used under visible light, respectively. Collectively, the accuracy, sensitivity, ease, economy, and flexibility of our strategy may reinforce the application of GFP in molecular biology.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Proteínas Fluorescentes Verdes , Peso Molecular , Proteínas Recombinantes de Fusión , beta-Galactosidasa , Western Blotting/métodos , Cromatografía de Afinidad , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.