RESUMEN
[This corrects the article DOI: 10.18632/oncotarget.18551.].
RESUMEN
Quantification of circulating tumor cells (CTCs) in blood samples from cancer patients is a non-invasive approach to monitoring the status of the disease. Most of the methods proposed in the recent years are phenomenological and rely on the use of antibodies labelled with fluorophores, magnetic particles, or immobilized on surfaces to capture the CTCs. Herein, we designed and optimized a method that employs a glucose analogue labelled with a fluorophore which takes advantage of the different metabolic pathways of cancer cells to discern them from normal ones. Notably, we demonstrate that fluorescence signal in tumor cells can be greatly maximized by applying hyperoxia conditions without damaging the cells. These results are demonstrated by means of confocal fluorescence and flow-cytometry measurements in peripheral blood mononuclear cells (PBMC) extracted after Ficoll of human blood samples and spiked with a known concentration of MCF-7 tumor cells.
RESUMEN
Metastatic cancer patients require a continuous monitoring during the sequential treatment cycles to carefully evaluate their disease evolution. Repetition of biopsies is very invasive and not always feasible. Herein, we design and demonstrate a 3D-flow focusing microfluidic device, where all optics are integrated into the chip, for the fluorescence quantification of CTCs in real samples. To test the chip performance, two cell membrane targets, the epithelial cell adhesion molecule, EpCAM, and the receptor tyrosine-protein kinase, HER2, are selected. The efficiency of the platform is demonstrated on cell lines and in a variety of healthy donors and metastatic-breast cancer patients.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Células Neoplásicas Circulantes/patología , Adulto , Anciano , Biomarcadores , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Imagen por Resonancia Magnética/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismoRESUMEN
The presence of a protein corona on various synthetic nanomaterials has been shown to strongly influence how they interact with cells. However, it is unclear if the protein corona also exists on protein particles, and if so, its role in particle-cell interactions. In this study, pure human serum albumin (HSA) particles were fabricated via mesoporous silica particle templating. Our data reveal that various serum proteins adsorbed on the particles, when exposed to human blood plasma, forming a corona. In human umbilical vein endothelial cells (HUVECs), the corona was shown to decrease particle binding to the cell membrane, increase the residence time of particles in early endosomes, and reduce the amount of internalized particles within the first hours of exposure to particles. These findings reveal important information regarding the mechanisms used by vascular endothelial cells to internalize protein-based particulate materials exposed to blood plasma. The ability to control the cellular recognition of these organic particles is expected to aid the advancement of HSA-based materials for intravenous drug delivery.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Nanoporos , Corona de Proteínas/química , Corona de Proteínas/metabolismo , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Humanos , Lisosomas/metabolismo , Transporte de ProteínasRESUMEN
Blood-based biomarkers (liquid biopsy) offer extremely valuable tools for the noninvasive diagnosis and monitoring of tumors. The protein c-MYC, a transcription factor that has been shown to be deregulated in up to 70% of human cancers, can be used as a robust proteomic signature for cancer. Herein, we developed a rapid, highly specific, and sensitive surface-enhanced Raman scattering (SERS) assay for the quantification of c-MYC in real blood samples. The sensing scheme relies on the use of specifically designed hybrid plasmonic materials and their bioderivatization with a selective peptidic receptor modified with a SERS transducer. Peptide/c-MYC recognition events translate into measurable alterations of the SERS spectra associated with a molecular reorientation of the transducer, in agreement with the surface selection rules. The efficiency of the sensor is demonstrated in cellular lines, healthy donors and a cancer patient.
RESUMEN
It is qualitatively demonstrated that the intracellular localization of particles depends on the way they are administered, their basic physicochemical properties, as well as on incubation time. For this purpose cells were exposed to fluorescently-labelled particles of different size under different exposure scenarios including incubation, microinjection, electroporation, and sonoporation. After co-exposure to cells the intracellular distribution of different particles was imaged with multicolor fluorescence microscopy. Qualitative co-localization analysis demonstrates, that different particles to which cells have been exposed in different ways did not automatically reside in the same compartment. As intracellular particle localization may influence potential toxic effects of particles on cells, studies attempting to unravel molecular mechanisms of toxicity should involve the determination of the intracellular particle distribution.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Macrófagos/efectos de los fármacos , Nanopartículas/toxicidad , Células A549 , Transporte Biológico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Electroporación , Colorantes Fluorescentes/química , Células HeLa , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Microinyecciones , Microscopía Fluorescente , Nanopartículas/química , Tamaño de la Partícula , Ondas UltrasónicasRESUMEN
Acidification of eukaryotic cell compartments is accomplished by vacuolar H+-ATPases (V-ATPases), large multisubunit complexes able to pump protons into the lumen of organelles or in the extracellular medium. V-ATPases are involved in a number of physiological cellular processes, and thus regulation of V-ATPase activity is of crucial importance for the cell. Indeed, dysfunction of V-ATPase or alterations of acidification have been recently recognized as key factors in a variety of human diseases. In this study, we applied capsule-based pH sensors and a real-time tracking method for investigating the role of the V1G1 subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V1G1. Second, we used fluorescent capsule-based pH sensors to monitor acidification before and during internalization by modified and control living cells. By using a simple real-time method for tracking capsule internalization, we were able to identify different capsule acidification levels with respect to each analyzed cell and to establish the kinetics for each. The intracellular pH measurements indicate a delay in acidification in either V1G1-overexpressing or V1G1-silenced cells compared to controls. Finally, in an independent set of experiments, we applied transmission electron microscopy and confocal fluorescence microscopy to further investigate the internalization of the capsules. Both analyses confirm that capsules are engulfed in acidic vesicular structures in modified and control cell lines. The use of capsule-based pH sensors allowed demonstration of the importance of the V1G1 subunit in V-ATPase activity concerning intravesicular acidification. We believe that the combined use of these pH-sensor system and such a real-time method for tracking their internalization path would contribute to systematically measure the proton concentration changes inside the endocytic compartments in various cell systems. This approach would provide fundamental information regarding molecular mechanisms and factors that regulate intracellular acidification, vesicular trafficking, and cytoskeletal reorganizations.
Asunto(s)
Endosomas/química , Endosomas/metabolismo , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Nanocápsulas/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Concentración de Iones de Hidrógeno , Imagen Molecular/métodosRESUMEN
Colloidal particles with fluorescence read-out are commonly used as sensors for the quantitative determination of ions. Calcium, for example, is a biologically highly relevant ion in signaling, and thus knowledge of its spatio-temporal distribution inside cells would offer important experimental data. However, the use of particle-based intracellular sensors for ion detection is not straightforward. Important associated problems involve delivery and intracellular location of particle-based fluorophores, crosstalk of the fluorescence read-out with pH, and spectral overlap of the emission spectra of different fluorophores. These potential problems are outlined and discussed here with selected experimental examples. Potential solutions are discussed and form a guideline for particle-based intracellular imaging of ions.
Asunto(s)
Técnicas Biosensibles , Calcio/química , Nanotecnología/métodos , Óptica y Fotónica , Benzoxazinas/química , Endocitosis , Colorantes Fluorescentes/química , Oro/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Iones , Nanopartículas del Metal/química , Microscopía Fluorescente , Tamaño de la Partícula , Péptidos/química , Polímeros/químicaRESUMEN
The efficiency of siRNA delivery is demonstrated to be improved by encapsulating the siRNA within a non-viral carrier based on layer-by-layer assembly of oppositely charged biodegradable and biocompatible polyelectrolytes. In comparison to other non-viral delivery vehicles such as polycation-based complexes, a smaller amount of siRNA was necessary to produce in vitro gene silencing as early as 20-30 h after incubation. Colloidal carriers based on assembled biodegradable polyelectrolytes offer several advantages, such as efficient intracellular delivery after endocytosis followed by release to the cytosol, as well as protection of the siRNA, which is crucial for its therapeutic activity.
Asunto(s)
Cápsulas , Silenciador del Gen/efectos de los fármacos , Vectores Genéticos/administración & dosificación , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Coloides , Composición de Medicamentos , Endocitosis/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Polietileneimina/toxicidadRESUMEN
The interfacing of colloidal nanoparticles with mammalian cells is now well into its second decade. In this review our goal is to highlight the more generally accepted concepts that we have gleaned from nearly twenty years of research. While details of these complex interactions strongly depend, amongst others, upon the specific properties of the nanoparticles used, the cell type, and their environmental conditions, a number of fundamental principles exist, which are outlined in this review.
RESUMEN
The concept of a long-term sensor for ion changes in the lysosome is presented. The sensor is made by layer-by-layer assembly of oppositely charged polyelectrolytes around ion-sensitive fluorophores, in this case for protons. The sensor is spontaneously incorporated by cells and resides over days in the lysosome. Intracellular changes of the concentration of protons upon cellular stimulation with pH-active agents are monitored by read-out of the sensor fluorescence at real time. With help of this sensor concept it is demonstrated that the different agents used (Monensin, Chloroquine, Bafilomycin A1, Amiloride) possessed different kinetics and mechanisms of action in affecting the intracellular pH values.