The cellular delivery of cell-impermeable and negatively-charged molecules remains a major challenge. The cyclic peptide [WR]5 containing alternative tryptophan (W) and arginine (R) residues showed significant improvement in the cellular uptake of anti-HIV drugs, doxorubicin, phosphopeptides, and oligonucleotides. We hypothesized that arginine residues on a cyclic ring conjugated with a hydrophobic linear chain of tryptophan residues in a hybrid peptide [RX]WY (X and Y = 5-7) can significantly improve the molecular transporter property versus [WR]5. Therefore, peptides containing R, W, and lysine (K) residues, namely [R5K]W5, [R5K]R6, [R6K]W5, [R6K]W6 ,[R5K]W7, [R7K]W5, and [R7K]W7 were synthesized using Fmoc solid-phase chemistry and cyclization. In vitro cytotoxicity of the peptides was evaluated by MTS assay using human leukemia (CCRF-CEM), ovarian cancer cell line (SK-OV-3), and normal kidney cell line (LLCPK). The synthesized peptides were not cytotoxic to normal (LLCPK) cells at a concentration of 25 μM. Among all the peptides, [R5K]W7 significantly improved the uptake of fluorescent-labeled phosphopeptide (F′-GpYEEI) by 48 fold and the cellular uptake of fluorescent-labeled emtricitabine (F′-FTC) and fluorescent-labeled stavudine (F′-d4T), respectively, by 27, and 36 times in CCRF-CEM cells, respectively, that was higher than that of [WR]5. The cellular uptake of fluorescent-labeled F′-[R5K]W7 was shown to be concentration- and time-dependent. Intracellular uptake of F′-[R5K]W7 in CCRF-CEM cells was still observed in the presence of sodium azide, suggesting that the cellular uptake was not completely ATP-dependent. Furthermore, F′-[R5K]W7 showed high cellular uptake in the presence of different endocytic inhibitors ruling out clathrin-mediated and caveolae-mediated endocytosis. [R5K]W7 was found to be significantly more efficient as a molecular transporter than [WR]5 and other hybrid peptides, suggesting that this peptide can be used for more efficient delivery of cell-impermeable biomolecules.

Amphiphilic cell-penetrating hybrid cyclic-linear peptides as a drug delivery system

256th National Meeting (2018)

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