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.
","abstract_html":"<p>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]<sub>5</sub> 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)<sub>5</sub>]K(RW)<sub>X</sub> (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).</p> <p><em>In vitro</em> 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.</p> <p>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)<sub>5</sub>K](RW)<sub>5</sub> (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]<sub>5</sub>.</p> <p>The cellular uptake of F′-[(RW)<sub>5</sub>K](RW)<sub>5</sub> 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)<sub>5</sub>K](RW)<sub>5</sub>) 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)<sub>5</sub>K](RW)<sub>5</sub> 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]<sub>5</sub> as molecular transporters of large molecules, such as a negatively charged phosphopeptide.</p>","abstract_has_math":false,"creators":["Khayyatnejad Shoushtari, Sorour"],"institution":null,"degree_name":"Master of Science (MS)","degree_level":"Thesis","degree_discipline":"Pharmaceutical Sciences","degree_department":null,"school":null,"contributors":["Keykavous Parang, Pharm.D., Ph.D.","Rakesh Tiwari, Ph.D.","Hamidreza Montazeri Aliabadi, Ph.D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-08-01T07:00:00Z","date_published":"2021-08-01T07:00:00Z","updated_at":"2026-07-24T01:38:16Z","subjects":["cell-penetrating peptide","cyclic-linear peptide","cellular uptake","drug delivery","Other Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/16","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Keykavous Parang, Pharm.D., Ph.D.","Rakesh Tiwari, Ph.D.","Hamidreza Montazeri Aliabadi, Ph.D."]},{"key":"dc:creator","label":"Author","values":["Khayyatnejad Shoushtari, Sorour"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2023-06-03T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MS)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["cell-penetrating peptide","cyclic-linear peptide","cellular uptake","drug delivery","Other Pharmacy and Pharmaceutical Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/16"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:source","label":"Dc Source","values":["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"]},{"key":"dc:title","label":"Title","values":["Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positive and Hydrophobic Residues as Molecular Transporters"]}]}],"canonical_facts":{"dc:contributor":["Keykavous Parang, Pharm.D., Ph.D.","Rakesh Tiwari, Ph.D.","Hamidreza Montazeri Aliabadi, Ph.D."],"dc:creator":["Khayyatnejad Shoushtari, Sorour"],"dc:date.available":["2023-06-03T07:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/16"],"dc:source":["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"],"dc:subject":["cell-penetrating peptide","cyclic-linear peptide","cellular uptake","drug delivery","Other Pharmacy and Pharmaceutical Sciences"],"dc:title":["Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positive and Hydrophobic Residues as Molecular Transporters"],"thesis:degree_discipline":["Pharmaceutical Sciences"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science (MS)"]},"updated_at":"2026-07-24T01:38:16Z"}