Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positive and Hydrophobic Residues as Molecular Transporters

Author

Sorour Khayyatnejad Shoushtari, Chapman UniversityFollow

Date of Award

Spring 8-2021

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Keykavous Parang, Pharm.D., Ph.D.

Second Advisor

Rakesh Tiwari, Ph.D.

Third Advisor

Hamidreza Montazeri Aliabadi, Ph.D.

Abstract

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.

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Recommended Citation

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