Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized and evaluated as molecular transporters and antimicrobial agents. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides. The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood) and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F′-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]5 significantly improved the uptake of F′-GpYEEI by approximately 110-folds after 3 h incubation. Confocal microscopy showed approximately 6-folds enhancement for the intracellular delivery of F′-GpYEEI in the presence of [DipR]5 for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F′-GpYEEI and [DipR]5 was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors, such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, which suggested that the uptake for [DipR]5 was not fully endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicate that [DipR]5 and Fluorescence-labeled [DipR]5 can act as a molecular transporter of several compounds, including the cell-impermeable phosphopeptide and siRNA specifically.

Peptides containing arginine, tryptophan, and diphenylalanine as molecular transporters

ACS Spring Meeting 2021

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