Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells

5 years ago

Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells

Shalondria Sears, Ewa P. Wojcikiewicz, Jing Ye, Patrick. J. Cosme, Andrew C. Terentis

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 ∼20 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 β-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.

Publisher URL: http://dx.doi.org/10.1021/acs.molpharmaceut.7b00601

DOI: 10.1021/acs.molpharmaceut.7b00601