Date of Award

Summer 8-2022

Document Type


Degree Name

Master of Science (MS)


Pharmaceutical Sciences

First Advisor

Keykavous Parang

Second Advisor

Rakesh Tiwari

Third Advisor

Hamidreza Montazeri Aliabadi


The cell membrane has polar and negative charged characteristics and separates the inside of cells from the outside environment, and only allows small molecules to go into the cell to protect them from toxic substances. However, cell membrane characteristics make it difficult for polar and negatively-charged molecules to interact and penetrate the membrane. The delivery of cell impermeable compounds, such as phosphopeptides and siRNA, across the cell membrane remains a challenge. Previous studies in Dr. Parang’s laboratory have shown that cyclic peptides composed of tryptophan (W) and arginine (R) amino acids [WR]5 have higher cellular uptake than the corresponding linear peptides and can act as molecular transporters. Herein we report the synthesis of linear and cyclic peptides containing alternate WW and RR residues and the evaluation of their cytotoxicity, cellular uptake, and confocal microscopy of peptides compared to [WR]5. The linear and cyclic peptides were synthesized, purified by high-performance liquid chromatography (HPLC), and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Then, cytotoxicity of peptides was determined in breast cancer (MDA-MB-231), ovarian cancer (SK-OV-3), leukemia (CCRF-CEM), and normal human kidney (HEK 293) cells after 48, 24, and 3 h incubation with various concentrations. The cytotoxicity showed the non-cytotoxic concentration was 5 μM. The cellular uptake (FACS) and confocal microscopy experiments were performed using MDA-MB-231 and SK-OV-3 cancer cells using a non-cytotoxic concentration of 5 μM of the peptides and 1 μM of fluorescence-labeled phosphopeptide (F′-GpYEEI), 760 nM of fluorescence-labeled siRNA (F′-siRNA), or 1 µM of fluorescence-labeled lamivudine (F′-3TC) with incubation for 3 h. A number of peptides were able to enhance the intracellular delivery of fluorescence-labeled cargoes significantly more than [WR]5. A comparison of the peptides showed that cyclic peptide [WWRR]5 (C4)was the most efficient molecular transporter. Confocal microscopy was consistent with the flow cytometry studies, as C4 was able to deliver F′-GpYEEI to the cytosol. Fluorescence-labeled C4 (F′-C4) was synthesized and demonstrated time- and concentration-dependent uptake, as shown by flow cytometry. Confocal microscopy showed the localization of the fluorescence-tagged peptide in the cytosol. The mechanistic studies in the presence of endocytosis inhibitors demonstrated that the endocytosis contributes to the cellular uptake. Moreover, F′-C4 had the lowest cellular uptake in the presence of chlorpromazine, indicating that the uptake was mostly dependent on the clathrin-mediated pathway. These data provide insight into the designing of the next generation of cell-penetrating peptides.

Creative Commons License

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

Available for download on Thursday, June 27, 2024