RESUMO
The development of QDs-based fluorescent bionanoprobe for cellular imaging fundamentally relies upon the precise knowledge of particle-cell interaction, optical properties of QDs inside and outside of the cell, movement of a particle in and out of the cell, and the fate of particle. We reported engineering and physicochemical characterization of water-dispersible Eu3+/Mn2+ co-doped ZnSe@ZnS core/shell QDs and studied their potential as a bionanoprobe for biomedical applications, evaluating their biocompatibility, fluorescence behaviour by CytoViva dual mode fluorescence imaging, time-dependent uptake, endocytosis and exocytosis in RAW 264.7 macrophages. The oxidation state and local atomic structure of the Eu dopant studied by X-ray absorption fine structure (XAFS) analysis manifested that the Eu3+ ions occupied sites in both ZnSe and ZnS lattices for the core/shell QDs. A novel approach was developed to relieve the excitation constraint of wide bandgap ZnSe by co-incorporation of Eu3+/Mn2+ codopants, enabling the QDs to be excited at a wide UV-visible range. The QDs displayed tunable emission colors by a gradual increase in Eu3+ concentration at a fixed amount of Mn2+, systematically enhancing the Mn2+ emission intensity via energy transfer from the Eu3+ to Mn2+ ion. The ZnSe:Eu3+/Mn2+@ZnS QDs presented high cell viability above 85% and induced no cell activation. The detailed analyses of QDs-treated cells by dual mode fluorescence CytoViva microscopy confirmed the systematic color-tunable fluorescence and its intensity enhances as a function of incubation time. The QDs were internalized by the cells predominantly via macropinocytosis and other lipid raft-mediated endocytic pathways, retaining an efficient amount for 24 h. The unique color tunability and consistent high intensity emission make these QDs useful for developing a multiplex fluorescent bionanoprobe, activatable in wide-visible region.
Assuntos
Corantes Fluorescentes/química , Pontos Quânticos/química , Animais , Európio/química , Európio/metabolismo , Európio/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Manganês/química , Manganês/metabolismo , Manganês/toxicidade , Camundongos , Microscopia de Fluorescência , Pontos Quânticos/metabolismo , Pontos Quânticos/toxicidade , Células RAW 264.7 , Compostos de Selênio/química , Compostos de Selênio/metabolismo , Compostos de Selênio/toxicidade , Sulfetos/química , Sulfetos/metabolismo , Sulfetos/toxicidade , Compostos de Zinco/química , Compostos de Zinco/metabolismo , Compostos de Zinco/toxicidadeRESUMO
Canthin-6-one alkaloids, which are present in plants of the genus Simaba, are natural compounds that are capable of acting as fluorescent probes. However, the chemical composition and fluorescent properties of most species of this genus have not been analyzed. The objective of this study was to characterize the fluorescent properties of an extract of S. bahiensis and identify the chemical entities responsible for these properties. In addition, the cell-labeling properties of the fluorescent dye from A and of the isolated compounds were characterized by confocal fluorescence microscopy and flow cytometry. One quassinoid and three fluorescent alkaloids were isolated from S. bahiensis, all compounds were identified by using NMR spectroscopy and high-resolution mass spectrometry. Staining experiments and HPLC-FL analysis shown that canthin-6-one alkaloids are the main green fluorescent compounds in the analyzed dyes. All compounds evaluated showed a cytoplasmic marker with a residence time of 24â h. The present study is the first to describe the presence of canthin-6-one alkaloids in S. bahiensis, in addition to demonstrating promising cell-labeling properties of fluorescent compounds from S. bahiensis with broad emission wavelengths.
Assuntos
Carbolinas/química , Corantes Fluorescentes/química , Alcaloides Indólicos/química , Simaroubaceae/química , Carbolinas/isolamento & purificação , Carbolinas/toxicidade , Corantes Fluorescentes/isolamento & purificação , Corantes Fluorescentes/toxicidade , Células Hep G2 , Humanos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/toxicidade , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Raízes de Plantas/químicaRESUMO
BACKGROUND: Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery. RESULTS: We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long ß-sheet breaker peptide that is able to recognize amyloid ß (Aß) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains. CONCLUSIONS: The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aß aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aß aggregates involved in the pathogenesis of the Alzheimer disease.
Assuntos
Doença de Alzheimer/diagnóstico , Amiloide/análise , Corantes Fluorescentes/química , Nanodiamantes/química , Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Animais , Benzotiazóis/química , Benzotiazóis/toxicidade , Biomarcadores/análise , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Corantes Fluorescentes/toxicidade , Humanos , Camundongos Transgênicos , Nanodiamantes/toxicidade , Agregados ProteicosRESUMO
Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safeTM, a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safeTM. For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safeTM. In conclusion, SYBR safeTM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.
Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Dano ao DNA/efeitos dos fármacos , Etídio/toxicidade , Corantes Fluorescentes/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
AIMS: There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. MAIN METHODS: The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. KEY FINDINGS: This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. SIGNIFICANCE: The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus.
Assuntos
Corantes Fluorescentes/metabolismo , Complexo de Golgi/metabolismo , Peptídeos Cíclicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/toxicidade , Células HCT116 , Humanos , Microscopia de Fluorescência , Peptídeos Cíclicos/toxicidade , Coloração e RotulagemRESUMO
Lightsticks are fishing gadgets that provide fluorescent lighting when two organic solutions are mixed. In NE Brazil, low-income coastal residents ignore their conventional use and collect lightsticks stranded on beaches. The lightstick solution is then used for various purposes, including direct human skin exposure. We assessed the reactions and possible cell damages on the skin of Wistar rats. Animals were exposed to lightstick contents, UV radiation and/or seawater. Lightstick exposure led to erythemas, oedemas and vesicles. Histopathologic alterations included proliferation of the epidermis and inflammatory infiltrates. In spite of the short time of experimentation (4 days), the rats exposed to the lightstick content alone and together with UV radiation and/or seawater provided evidence of irritation/alteration reactions that may evolve into skin cancer. Our results demonstrated a few of the potential problems associated with lightstick dumping into the ocean and highlight the need for further investigations about this new type of marine pollutant.
Assuntos
Dermatite Irritante/etiologia , Corantes Fluorescentes/toxicidade , Água do Mar/efeitos adversos , Pele , Raios Ultravioleta/efeitos adversos , Poluentes Químicos da Água/toxicidade , Animais , Praias , Brasil , Dermatite Irritante/patologia , Pesqueiros , Masculino , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/patologia , Pele/efeitos da radiação , Testes de Irritação da PeleRESUMO
Excited fluorophores produce reactive oxygen species that are toxic toward many live cells (phototoxicity) and accelerate bleaching of the fluorophores during the course of extended or repeated measurements (photobleaching). We recently developed an illumination system for fluorescence microscopy using a high power light-emitting diode (LED), which can emit short pulses of light (0.5-2 ms) to excite fluorophores. This system minimizes illumination time, thus reducing phototoxicity and photobleaching artifacts. To demonstrate the usefulness of the new system, we compared images of human sperm loaded with various fluorescent indicators and excited with either a conventional mercury lamp as a continuous excitation light source or the LED as a source of pulsed illumination. We found that sperm motility decreased rapidly and photobleaching was relatively rapid under continuous illumination, whereas under pulsed LED illumination, motility was maintained and photobleaching was much reduced. Therefore, fluorescence microscopy using LED-based pulsed illumination offers significant advantages for long-term live cell imaging, reducing the degree of phototoxicity, and extending the effective lifetime of fluorophores.
Assuntos
Corantes Fluorescentes/toxicidade , Luz , Fotodegradação/efeitos da radiação , Espermatozoides/fisiologia , Estroboscopia , Cálcio/metabolismo , Humanos , Cinética , Masculino , Sêmen/citologiaRESUMO
Recent studies from our laboratory provided evidence that part of the carcinogenic effects of ethanol consumption might be related to its in situ metabolism at cytosolic and microsomal levels, to the mutagen acetaldehyde and to hydroxyl and 1-hydroxyethyl radicals. In this work, we report on our experiments where Sprague-Dawley female rats were exposed to the standard Lieber & De Carli diet for 28 days. We observed: the induction of the (xanthineoxidoreductase mediated) cytosolic and microsomal (lipoxygenase mediated) pathways of ethanol metabolism; promotion of oxidative stress as shown by increased formation of lipid hydroperoxides; delay in the t-butylhydroperoxide induced chemiluminiscence, and a significant decrease in protein sulfhydryls. In addition, the epithelial cells showed ultrastructural alterations consisting of markedly irregular nuclei, with frequent invaginations at the level of the nuclear envelope, condensation of chromatin around the inner nuclear membrane, and marked dilatation of the nuclear pores showing filamentous material exiting to the cytoplasm. In conclusion, the presence in mammary epithelial cells of cytosolic and microsomal pathways of ethanol bioactivation to carcinogenic and to tumorigenic metabolites might play a role in alcohol promotion of breast cancer.
Assuntos
Acetaldeído/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Neoplasias da Mama/induzido quimicamente , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Glândulas Mamárias Animais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Depressores do Sistema Nervoso Central/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Dieta , Etanol/metabolismo , Feminino , Corantes Fluorescentes/toxicidade , Radicais Livres/metabolismo , Humanos , Imuno-Histoquímica , Glândulas Mamárias Animais/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Fenóis , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/metabolismo , Sulfóxidos , Xantina Oxidase/metabolismo , Xilenos/toxicidadeAssuntos
Ciclídeos/fisiologia , Corantes Fluorescentes/farmacocinética , Corantes Fluorescentes/toxicidade , Pirenos/farmacocinética , Pirenos/toxicidade , Poluentes Químicos da Água/farmacocinética , Poluentes Químicos da Água/toxicidade , Animais , Corantes Fluorescentes/administração & dosagem , Injeções Intraperitoneais , Pirenos/administração & dosagem , Medição de Risco , Poluentes Químicos da Água/administração & dosagemRESUMO
Peripheral nerve ultrastructure was assessed after single or multiple local injections of the intercalating dye ethidium bromide. Thirty-four adult Wistar rats of both sexes were divided into five groups and maintained in a controlled environment with rat chow and water ad libitum throughout the experiment. The experimental animals were injected with 1 microl of 0.1% ethidium bromide in 0.9% saline into the central third of the left sciatic nerve 1 (group 1), 2 (group 2), 4 (group 3), 6 (group 4) or 8 (group 5) times. In groups 2 to 5 the injections were made at 28-day intervals. Control animals received the same amount of 0.9% saline. The animals were killed at different times after injection: group 1 at 7 days (2 rats) and 15 days (2 rats); for groups 2, 3, 4 and 5, all rats were killed 10 days after the last injection and the lesions were investigated by light and transmission electron microscopy. In the acute lesions, intoxicated Schwann cells showed a vacuolated cytoplasm and separation of the sheaths from the axon. Myelin sheaths underwent progressive vesiculation and subsequent segmental demyelination. Myelin debris were withdrawn by macrophages and remyelination by Schwann cells was prominent. With the increase in the number of injections collagen fibers also increased in number and progressively enveloped smaller numbers of remyelinated axons composing new fascicles. Wallerian degeneration of fibers apparently not affected by ethidium bromide was more intense in the nerves from groups 4 and 5. The peripheral nerve repairs itself after demyelinating challenges with a profusion of collagen fibers and new fasciculations. This experimental model is valid to mimic recurrent demyelinating neuropathies.