RESUMEN
Cell-penetrating peptides (CPPs) are promising vectors for the intracellular delivery of a variety of membrane-impermeable bioactive compounds. The mechanisms by which CPPs cross the cell membrane, and the effects that CPPs may have on cell function, still remain to be fully clarified. In this work, we employed confocal Raman microscopy (CRM) and atomic force microscopy (AFM) to study the infiltration and physiological effects of the amphipathic CPP transportan (Tp) on the metastatic melanoma cell line SK-Mel-2. CRM enabled the detection of label-free Tp within the cells. Raman maps of live cells revealed rapid entry (within 5 min) and widespread distribution of the peptide throughout the cytoplasm and the presence of the peptide within the nucleus after â¼20 min. Principal component analysis of the CRM data collected from Tp-treated and untreated cells showed that Tp Raman bands were not positively correlated with lipid Raman bands, indicating that Tp entered the cells via a nonendocytic mechanism. Analysis of intracellularly recovered Tp by mass spectrometry showed that Tp remained intact in SK-Mel-2 cells for up to 24 h. The Raman spectroscopic data also showed that, although Tp was predominantly unstructured (random coil) in aqueous solution, it accumulated to high densities within the cells with mostly ß-sheet and α-helical structures. AFM was employed to measure the effect of Tp treatment on cell stiffness. These data showed that Tp induced a significant increase in cell stiffness within the first hour of treatment, which was partially abated after 2 h. It is hypothesized that the increase in cell stiffness was the result of cytoskeletal changes triggered by Tp.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Portadores de Fármacos/farmacología , Galanina/farmacología , Microscopía Intravital/métodos , Proteínas Recombinantes de Fusión/farmacología , Espectrometría Raman/métodos , Venenos de Avispas/farmacología , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Humanos , Melanoma/tratamiento farmacológico , Microscopía de Fuerza Atómica , Microscopía Confocal/métodos , Análisis de Componente PrincipalRESUMEN
The addition of carbamate nitrogen to a non-conjugated carbon-carbon triple bond is catalyzed by an ammonium salt leading to a cyclic product. Studies in homogeneous systems suggest that the ammonium agent facilitates nitrogen-carbon bond formation through a cation-π interaction with the alkyne unit that, for the first time, is directly observed by Raman spectroscopy.
Asunto(s)
Alquinos/química , Compuestos de Amonio/química , Catálisis , Cationes , Ciclización , Espectrometría Raman/métodosRESUMEN
Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (â¼5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND-biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND-peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to.
Asunto(s)
Colágeno , Nanodiamantes/química , Péptidos , Animales , Células CHO , Adhesión Celular/efectos de los fármacos , Colágeno/química , Colágeno/farmacología , Cricetinae , Cricetulus , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Estructura Secundaria de ProteínaRESUMEN
We identify distinct site-specific dynamics over the time course of Aß1-23 amyloid formation by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. Our results also suggest the key role of an edge-to-face aromatic interaction in the conformational conversion to form and stabilize ß-sheet structure.