Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells Patrick. J. Cosme, †,§ Jing Ye, †,§ Shalondria Sears, ‡ Ewa P. Wojcikiewicz, ‡ and Andrew C. Terentis* ,† † Department of Chemistry and Biochemistry and ‡ Department of Biomedical Science, Florida Atlantic University, Boca Raton, Florida 33431, United States * S Supporting Information ABSTRACT: Cell-penetrating peptides (CPPs) are promis- ing vectors for the intracellular delivery of a variety of membrane-impermeable bioactive compounds. The mecha- nisms 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. KEYWORDS: transportan, cell-penetrating peptide, confocal Raman microscopy, atomic force microscopy ■ INTRODUCTION Cell-penetrating peptides (CPPs) are peptides that are able to cross the plasma membrane of most cell types with high efficiency and low cytotoxicity. Interest in CPPs stems from their potential therapeutic value, which includes attaching CPPs to a wide variety of bioactive compounds to improve cellular delivery. 1-3 Although they have been identified since the 1980s, the structural features that determine CPP function and the mechanisms by which CPPs cross the cell membrane still remain unclear. We now know that a net positive charge is a defining characteristic of many CPPs, as is the ability to cross the cell membrane via both active (endocytic) and passive (nonendocytic) transport mechanisms. Critical parameters that appear to influence the mechanism of uptake include the extracellular peptide concentration, peptide sequence, the cell line and its membrane components, whether cargo is conjugated to the CPP, the nature of the cargo, and the nature of the peptide-cargo link. 1,3-6 For some CPPs, an apparent threshold for the extracellular concentration has been observed, generally in the low micromolar range, where endocytic pathways appear to predominate below the concentration threshold, whereas nonendocytic pathways are more active above the threshold. 3,4 Transportan (Tp) is a chimeric CPP first synthesized and studied in the mid 1990s. 7 Tp is formed by the conjugation of two smaller peptides, a fragment of the neuropeptide galanin forming the N-terminal side and the wasp venom peptide mastoparan forming the C-terminal side, with both fragments joined by a central lysine residue. The full 27 amino acid sequence, GWTLNSAGYLLGKINLKALAALAKKIL, classifies it as a primary amphipathic peptide with a net positive charge. 3 Biotinylated Tp (biotin-Tp) was imaged within fixed epithelial cells by fluorescence microscopy employing a fluorescent avidin probe, showing that biotin-Tp was localized in the nucleus and nuclear membrane and also diffusely throughout the cytoplasm after 1 h incubation with 10 μM of peptide at 37 °C. 8 In earlier work, biotin-Tp was detectable in the plasma membrane after 1 min, ER and Golgi complex membranes in the next 2-3 min, and in the nuclear membrane after 15 min incubation. 9 The speed of cell entry and impotency of endocytosis inhibitors suggested a nonendocytic uptake mechanism. 8 On the other Received: July 12, 2017 Revised: November 26, 2017 Accepted: February 4, 2018 Published: February 5, 2018 Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.molpharmaceut.7b00601 Mol. Pharmaceutics XXXX, XXX, XXX-XXX