RESUMEN
Free radicals are crucial indicators for stress and appear in all kinds of pathogenic conditions, including cancer, cardiovascular diseases, and infection. However, they are difficult to detect due to their reactivity and low abundance. We use relaxometry for the detection of radicals with subcellular resolution. This method is based on a fluorescent defect in a diamond, which changes its optical properties on the basis of the magnetic surroundings. This technique allows nanoscale MRI with unprecedented sensitivity and spatial resolution. Recently, this technique was used inside living cells from a cell line. Cell lines differ in terms of endocytic capability and radical production from primary cells derived from patients. Here we provide the first measurements of phagocytic radical production by the NADPH oxidase (NOX2) in primary dendritic cells from healthy donors. The radical production of these cells differs greatly between donors. We investigated the cell response to stimulation or inhibition.
Asunto(s)
Nanodiamantes , Células Dendríticas , Diamante , Radicales Libres , Humanos , Magnetismo , Nanodiamantes/químicaRESUMEN
The utilization of nanoparticles for the intracellular delivery of theranostic agents faces one substantial limitation. Sequestration in intracellular vesicles prevents them from reaching the desired location in the cytoplasm or nucleus to deliver their cargo. We investigated whether three different cell-penetrating peptides (CPPs), namely, octa-arginine R8, polyhistidine KH27K and histidine-rich LAH4, could promote cytosolic and/or nuclear transfer of unique model nanoparticles-pseudovirions derived from murine polyomavirus. Two types of CPP-modified pseudovirions that carry the luciferase reporter gene were created: VirPorters-IN with CPPs genetically attached to the capsid interior and VirPorters-EX with CPPs noncovalently associated with the capsid exterior. We tested their transduction ability by luciferase assay and monitored their presence in subcellular fractions. Our results confirmed the overall effect of CPPs on the intracellular destination of the particles and suggested that KH27K has the potential to improve the cytosolic release of pseudovirions. None of the VirPorters caused endomembrane damage detectable by the Galectin-3 assay. Remarkably, a noncovalent modification was required to promote high transduction of the reporter gene and cytosolic delivery of pseudovirions mediated by LAH4. Together, CPPs in different arrangements have demonstrated their potential to improve pseudovirion invasion into cells, and these findings could be useful for the development of other nanoparticle-based delivery systems.
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Péptidos de Penetración Celular , Animales , Bioensayo , Cationes , Citosol , Histidina , RatonesRESUMEN
Understanding virus-host interactions is crucial for vaccine development. This study investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100 nm FNDs with rA27(aa 21-84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein rDA27(aa 33-84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including rA27(aa 21-84)-FND, rDA27(aa 33-84)-FND, and hybrid rA27(aa 21-84)/rDA27(aa 33-84)-FND. The specificity of these viral protein-FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface. Our studies provide a new nanoparticle-based platform not only to target specific cell types but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGs-associated proteins on cell membrane.
Asunto(s)
Colorantes Fluorescentes/química , Glicosaminoglicanos/análisis , Nanodiamantes/química , Imagen Óptica/métodos , Proteínas del Envoltorio Viral/química , Animales , Línea Celular , Citometría de Flujo , Células HeLa , Humanos , Ratones , Microscopía FluorescenteRESUMEN
Recently discovered tunneling nanotubes (TNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. Intercellular transport is related to many diseases and nanotubes are potentially useful as drug-delivery channels for cancer therapy. Here, we apply fluorescent nanodiamond (FND) as a photostable tracker, as well as a protein carrier, to illustrate the transport events in TNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, are first coated on 100-nm FNDs by physical adsorption and then single-particle tracking of the bioconjugates in the transient membrane connections is carried out by fluorescence microscopy. Stop-and-go and to-and-fro motions mediated by molecular motors are found for the active transport of protein-loaded FNDs trapped in the endosomal vehicles of human embryonic kidney cells (HEK293T). Quantitative analysis of the heterotypical transport between HEK293T and SH-SY5Y neuroblastoma cells by flow cytometry confirm the formation of open-ended nanotubes between them, despite that their TNTs differ in structural components. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level.