Date of Award
Master of Science (MS)
Keykavous Parang, Pharm.D., Ph.D.
Rakesh Tiwari, Ph.D.
Hamidreza Montazeri Aliabadi, Ph.D.
The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Amphiphilic cyclic peptides containing alternative arginine and tryptophan such as [WR]5 have been shown to enhance the transport of cargo molecules across the cell membrane. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side-chain [(RW)5]K(RW)X (X=1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. Final compounds were purified by a reversed-phase HPLC (High Performance Liquid Chromatography). The chemical structures of the final products were confirmed by MALDI-TOF (Matrix Assisted Laser Desorption/Ionization).
In vitro cytotoxicity of the peptides was evaluated in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) using the MTS assay. The peptides did not exhibit any significant cytotoxicity at the concentration of 10 μM in CCRF-CEM, HEK-293, MDA-MB-231, and SK-OV-3 cells after 3 h incubation.
The cellular uptake of a fluorescence-labeled phosphopeptide (F′-GpYEEI) and anti-HIV drugs (lamivudine (F′-3TC), emtricitabine (F′-FTC), Stavudine (F′-d4T)), where F′ is carboxyfluorescein, were measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 μM) was the most efficient transporter and improved the cellular uptake of F′-GpYEEI (2 μM) by 18 and 11-fold in CCRF-CEM and SK-OV-3, respectively, when compared with F′-GpYEEI alone and in the presence of [WR]5.
The cellular uptake of F′-[(RW)5K](RW)5 was increased in a time- and concentration-dependent manner. The FACS (fluorescence-activated cell sorting) analysis and confocal microscopy results indicate that the cellular uptake of fluorescent-labeled peptide (F'-[(RW)5K](RW)5) was partially inhibited by chlorpromazine endocytosis inhibitors after 3 h incubation in MDA-MB-231 cells, which suggests the partial uptake through the clathrin-mediated endocytosis pathway. Intracellular localization (mostly in the cytosol) of F'-[(RW)5K](RW)5 after 3 h incubation in MDA-MB-231 cells was confirmed by confocal microscopy. These data suggest the potential of this series of hybrid cyclic-linear peptides compared to [WR]5 as molecular transporters of large molecules, such as a negatively charged phosphopeptide.
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Khayyatnejad Shoushtari, S. Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positive and Hydrophobic Residues as Molecular Transporters. [master’s thesis]. Irvine, CA: Chapman University; 2021. https://doi.org/10.36837/chapman.000284
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