RESUMEN
Probing the dynamics and quantifying the activities of intracellular protein kinases that coordinate cell growth and division and constitute biomarkers and pharmacological targets in hyperproliferative and pathological disorders remain a challenging task. Here engineering and characterization of a nanobiosensor of the mitotic kinase CDK1, through multifunctionalization of carbon nanotubes with a CDK1-specific fluorescent peptide reporter, are described. This original reporter of CDK1 activity combines the sensitivity of a fluorescent biosensor with the unique physico-chemical and biological properties of nanotubes for multifunctionalization and efficient intracellular penetration. The functional versatility of this nanobiosensor enables implementation to quantify CDK1 activity in a sensitive and dose-dependent fashion in complex biological environments in vitro, to monitor endogenous kinase in living cells and directly within tumor xenografts in mice by fluorescence imaging, thanks to a ratiometric quantification strategy accounting for response relative to concentration in space and in time.
Asunto(s)
Proteína Quinasa CDC2 , Nanotubos de Carbono , Neoplasias Experimentales/enzimología , Animales , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Humanos , Ratones , FosforilaciónRESUMEN
Luminescent ZnO nanocrystals were synthesized by basic hydrolysis of Zn(OAc)(2) in the presence of oleic acid and then functionalized with (poly)aminotrimethoxysilanes in the presence of tetramethylammonium hydroxide to render the QDs water-dispersible. The highest photoluminescence quantum yield (17%) was achieved using N(1)-(2-aminoethyl)-N(2)-[3-(trimethoxysilyl)propyl]-1,2-ethanediamine as surface ligand. Transmission electron microscopy and powder x-ray diffraction showed highly crystalline materials with a ZnO nanoparticle diameter of about 4 nm. The cytotoxicity of the different siloxane-capped ZnO QDs towards growing Escherichia coli bacterial cells was evaluated in MOPS-minimal medium. Although concentrations of 5 mM in QDs caused a complete growth arrest in E. coli, siloxane-capped ZnO QDs appeared weakly toxic at lower doses (0.5 or 1 mM). The concentration of bioavailable Zn (2+) ions leaked from ZnO QDs was evaluated using the biosensor bacteria Cupriavidus metallidurans AE1433. The results obtained clearly demonstrate that concentrations of bioavailable Zn(2+) are too low to explain the inhibitory effects of the ZnO QDs against bacteria cells at 1 mM and that the siloxane shell prevents ZnO QDs from dissolution contrary to uncapped ZnO nanoparticles. Because of their low cytotoxicity, good biocompatibility, low cost and large number of functional amine end groups, which makes them easy to tailor for end-user purposes, siloxane-capped ZnO QDs offer a high potential as fluorescent probes and as biosensors.
Asunto(s)
Nanopartículas/química , Puntos Cuánticos , Silanos/química , Óxido de Zinc/química , Absorción , Técnicas Biosensibles , Proliferación Celular/efectos de los fármacos , Cupriavidus/metabolismo , Escherichia coli/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Ácido Oléico/química , Silanos/farmacología , Siloxanos/química , Siloxanos/farmacología , Zinc/química , Zinc/farmacología , Óxido de Zinc/farmacologíaRESUMEN
Nanomaterials possess unique features which make them particularly attractive for biosensing applications. In particular, carbon nanotubes (CNTs) can serve as scaffolds for immobilization of biomolecules at their surface, and combine several exceptional physical, chemical, electrical, and optical characteristics properties which make them one of the best suited materials for the transduction of signals associated with the recognition of analytes, metabolites, or disease biomarkers. Here we provide a comprehensive review on these carbon nanostructures, in which we describe their structural and physical properties, functionalization and cellular uptake, biocompatibility, and toxicity issues. We further review historical developments in the field of biosensors, and describe the different types of biosensors which have been developed over time, with specific focus on CNT-conjugates engineered for biosensing applications, and in particular detection of cancer biomarkers.
RESUMEN
Infections associated with implanted medical devices are a major cause of nosocomial infections, with serious medical and economic repercussions. A variety of silver-containing coatings have been proposed to decrease the risk of infection by hindering bacterial adhesion and biofilm formation. However, the therapeutic range of silver is relatively narrow and it is important to minimize the amount of silver in the coatings, in order to keep sufficient antibacterial activity without inducing cytotoxicity. In this study, the antibacterial efficiency and biocompatibility of nanocoatings with minimal silver loading (â¼0.65 nmol cm(-2)) was evaluated in vitro and in vivo. Titanium substrates were coated by grafting mercaptododecylphosphonic acid (MDPA) monolayers followed by post-reaction with AgNO3. The MDPA/AgNO3 nanocoatings significantly inhibited Escherichia coli and Staphylococcus epidermidis adhesion and biofilm formation in vitro, while allowing attachment and proliferation of MC3T3-E1 preosteoblasts. Moreover, osteogenic differentiation of MC3T3 cells and murine mesenchymal stem cells was not affected by the nanocoatings. Sterilization by ethylene oxide did not alter the antibacterial activity and biocompatibility of the nanocoatings. After subcutaneous implantation of the materials in mice, we demonstrated that MDPA/AgNO3 nanocoatings exhibit significant antibacterial activity and excellent biocompatibility, both in vitro and in vivo, after postoperative seeding with S. epidermidis. These results confirm the interest of coating strategies involving subnanomolar amounts of silver exposed at the extreme surface for preventing bacterial adhesion and biofilm formation on metallic or ceramic medical devices without compromising their biocompatibility.
Asunto(s)
Antibacterianos/farmacología , Materiales Biocompatibles/farmacología , Organofosfonatos/farmacología , Plata/farmacología , Titanio/farmacología , Animales , Adhesión Bacteriana/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Escherichia coli/efectos de los fármacos , Óxido de Etileno/farmacología , Iones , Ratones Endogámicos C57BL , Ratones SCID , Pruebas de Sensibilidad Microbiana , Osteogénesis/efectos de los fármacos , Espectroscopía de Fotoelectrones , Soluciones , Staphylococcus epidermidis/efectos de los fármacos , EsterilizaciónRESUMEN
The synthesis and characterization of superparamagnetic iron(iii) oxide nanowires confined within double-walled carbon nanotubes by capillary filling with a melted precursor (iron iodide) followed by thermal treatment is reported for the first time.