RESUMO
Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 µM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.
Assuntos
Coproporfirinas/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Compostos Organoplatínicos/química , Oxigênio/análise , Animais , Técnicas Biossensoriais , Calibragem , Sobrevivência Celular , Corantes Fluorescentes/síntese química , Humanos , Medições Luminescentes , Sondas Moleculares/síntese química , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Oxigênio/metabolismo , Ratos , Sensibilidade e Especificidade , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O(2)-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O(2) monitoring in live cells and in tissue, and an alternative to the existing O(2) probes which require facilitated transport into the cell.
Assuntos
Corantes Fluorescentes/química , Medições Luminescentes/métodos , Oxigênio/análise , Peptídeos/química , Porfirinas/química , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais/métodos , Hipóxia Celular , Linhagem Celular Tumoral , Células HCT116 , Células HeLa , Células Hep G2 , Humanos , Células PC12 , Peptídeos/metabolismo , Platina/química , RatosRESUMO
Phosphorescent platinum(II) coproporphyrin label (PtCP) is evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labeled with a new maleimide derivative of PtCP and with the dark quencher dabcyl. Following photophysical characterization, the quenched substrate was employed in cleavage assays for caspase-3 using Jurkat and HL60 cell lines treated with proapoptotic stimuli performed on a commercial plate reader. Dose-response and time course assays for the drug camptothecin were obtained for comparison with conventional fluorometric detection.
