Abstract
Linear (HR)n and cyclic [HR]n peptides (n = 4,5) containing alternate arginine and histidine residues were synthesized. The peptides showed 0–15% cytotoxicity at 5–100 µM in human ovarian adenocarcinoma (SK-OV-3) cells while they exhibited 0–12% toxicity in human leukemia cancer cell line (CCRF-CEM). Among all peptides, cyclic [HR]4 peptide was able to improve the delivery of a cell impermeable fluorescence-labeled phosphopeptide by two-fold. Fatty acids of different alkyl chain length were attached at the N-terminal of the linear peptide (HR)4 to improve the molecular transporter property. Addition of fatty acyl chains was expected to help with the permeation of the peptides through the cell membrane. Thus, we synthesized seven fatty acyl derivatives of the linear (HR)4 peptide. The peptides were synthesized using Fmoc/tBu solid phase peptide chemistry, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization (MALDI) spectrometry. The fatty acyl peptides containing C8, C12, C14, and C18 alkyl chain did not show cytotoxicity on SK-OV-3 or CCRF-CEM cell lines up to 50 µM concentration; however, at higher concentration (100 µM), they showed mild cytotoxicity. For example, C16-(HR)4 was also found to reduce the proliferation of SK-OV-3 cells by 11% at 50 µM and C20-(HR)4 showed mild toxicity at 10 µM, reducing the proliferation of SK-OV-3 cells by 21%. Increase in the length of alkyl chain showed cytotoxicity to the cell lines. C20-(HR)4 peptide showed better efficiency in translocation of F′-GpYEEI to SK-OV-3 than the phosphopeptide alone. Further investigation of C20-(HR)4 peptide efficacy showed that the peptide could deliver doxorubicin and epirubicin into SK-OV-3 and also improved the drug antiproliferative ability. These studies provided insights into understanding the structural requirements for optimal cellular delivery of the fatty acyl-(HR)4 peptide conjugates.
Highlights
Cell-penetrating peptides (CPPs) known as protein transduction domains (PTDs), are a group of short peptides with up to 25–30 amino acids that are capable of penetrating the cell membrane.CPPs are characterized by their high loading capacity, high transduction efficiency and rapidMolecules 2018, 23, 1590; doi:10.3390/molecules23071590 www.mdpi.com/journal/moleculesMolecules 2018, 23, 1590 transduction rate [1,2]
We explored the combination of attaching different hydrophobic amino acids and charged residues with or without fatty acids to get homochiral CPPs as molecular transporters named [WR]4 and [WR]5 [2,20,21,22,23,24]
In the frame work of developing unique CPPs to explore their application of molecular transporter properties and the targeting of cargos to cancer cells, we aimed to develop novel CPPs with alternating arginine and histidine amino acids to evaluate their ability to translocate various biomolecular cargoes of different molecular size and hydrophobic nature to the cancer cells
Summary
Cell-penetrating peptides (CPPs) known as protein transduction domains (PTDs), are a group of short peptides with up to 25–30 amino acids that are capable of penetrating the cell membrane. Molecules 2018, 23, 1590 transduction rate [1,2] They are considered as good candidates for translocation of bioactive macromolecules, such as proteins, nucleic acids, peptides, inorganic particles and liposomes into cells [3,4,5,6]. Other classification is based on the origin of CPPs, dividing them into protein-derived. Two major routes for CPPs cellular internalization have been proposed: direct membrane translocation via pore-opening mechanism and endocytosis-mediated pathway, which divides into clathrin-dependent endocytosis, actin-dependent and caveolae-dependent micropinocytosis pathways [10]. The endosomal entrapment, potential cytotoxicity, immunogenicity and the lack of selectivity are the main obstacles that hinder the wide use of CPP’s in the fields of imaging, drug delivery, and treatment [11]
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