Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides.
The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F´-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]5 significantly improved the uptake of F´-GpYEEI, by approximately 110-folds after 3 h incubation. Confocal microscopy demonstrated the improved delivery of F´-GpYEEI (6-folds), which was mostly localized in the cytosol, in the presence of [DipR]5 for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F´-GpYEEI and [DipR]5 was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin suggesting that the uptake for [DipR]5 may not be endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicates that [DipR]5 and Fluorescence-labeled [DipR]5 can act as a molecular transporter of several compounds including the cell-impermeable phosphopeptide and siRNA specifically.
The antibacterial activities of peptides were evaluated against pathogens. The minimum inhibitory concentrations (MIC) of peptides were determined by micro-broth dilution protocol against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. [DipR]5 and((DipR)2(WR)3) showed promising MIC values of 0.39-6.25 µM against Gram-positive bacteria strains and 3.13-25 µM against Gram-negative bacteria strains.
","abstract_html":"<p>Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides.</p> <p>The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F´-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]<sub>5</sub> significantly improved the uptake of F´-GpYEEI, by approximately 110-folds after 3 h incubation. Confocal microscopy demonstrated the improved delivery of F´-GpYEEI (6-folds), which was mostly localized in the cytosol, in the presence of [DipR]<sub>5</sub> for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F´-GpYEEI and [DipR]<sub>5</sub> was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin suggesting that the uptake for [DipR]<sub>5</sub> may not be endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicates that [DipR]<sub>5 </sub>and Fluorescence-labeled [DipR]<sub>5</sub> can act as a molecular transporter of several compounds including the cell-impermeable phosphopeptide and siRNA specifically.</p> <p>The antibacterial activities of peptides were evaluated against pathogens. The minimum inhibitory concentrations (MIC) of peptides were determined by micro-broth dilution protocol against <em>Methicillin-Resistant</em> <em>Staphylococcus aureus</em>, <em>Klebsiella pneumoniae</em>, <em>Pseudomonas aeruginosa</em>, <em>Escherichia coli</em>, <em>Staphylococcus aureus</em>, <em>Enterococcus faecium</em>, <em>Enterococcus faecalis</em>, <em>Streptococcus pneumoniae</em>, and <em>Bacillus subtilis</em>. [DipR]<sub>5 </sub>and((DipR)<sub>2</sub>(WR)<sub>3</sub>) showed promising MIC values of 0.39-6.25 µM against Gram-positive bacteria strains and 3.13-25 µM against Gram-negative bacteria strains.</p>","abstract_has_math":false,"creators":["Salehi, David"],"institution":null,"degree_name":"Master of Science (MS)","degree_level":"Thesis","degree_discipline":"Pharmaceutical Sciences","degree_department":null,"school":null,"contributors":["Dr. Keykavous Parang","Dr. Rakesh Kumar Tiwari","Dr. Simin Rahighi"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-01-01T08:00:00Z","date_published":"2021-01-01T08:00:00Z","updated_at":"2026-07-24T01:38:16Z","subjects":["Cellular Uptake","Antimicrobial peptides","Nanoparticles","Diphenylalanine","Cancer and bacterial Infections","Gene Therapy","Amino Acids, Peptides, and Proteins","Bacterial Infections and Mycoses","Cancer Biology","Chemicals and Drugs","Medicinal and Pharmaceutical Chemistry","Medicinal Chemistry and Pharmaceutics","Nanomedicine","Nanotechnology","Nucleic Acids, Nucleotides, and Nucleosides","Other Pharmacology, Toxicology and Environmental Health","Other Pharmacy and Pharmaceutical Sciences","Pharmaceutical Preparations","Pharmaceutics and Drug Design","Pharmacology","Pharmacology, Toxicology and Environmental Health","Pharmacy and Pharmaceutical Sciences","Toxicology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/15","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Dr. Keykavous Parang","Dr. Rakesh Kumar Tiwari","Dr. Simin Rahighi"]},{"key":"dc:creator","label":"Author","values":["Salehi, David"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2023-01-20T08:00:00Z"]},{"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":["Cellular Uptake","Antimicrobial peptides","Nanoparticles","Diphenylalanine","Cancer and bacterial Infections","Gene Therapy","Amino Acids, Peptides, and Proteins","Bacterial Infections and Mycoses","Cancer Biology","Chemicals and Drugs","Medicinal and Pharmaceutical Chemistry","Medicinal Chemistry and Pharmaceutics","Nanomedicine","Nanotechnology","Nucleic Acids, Nucleotides, and Nucleosides","Other Pharmacology, Toxicology and Environmental Health","Other Pharmacy and Pharmaceutical Sciences","Pharmaceutical Preparations","Pharmaceutics and Drug Design","Pharmacology","Pharmacology, Toxicology and Environmental Health","Pharmacy and Pharmaceutical Sciences","Toxicology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/15"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides.
The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F´-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]5 significantly improved the uptake of F´-GpYEEI, by approximately 110-folds after 3 h incubation. Confocal microscopy demonstrated the improved delivery of F´-GpYEEI (6-folds), which was mostly localized in the cytosol, in the presence of [DipR]5 for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F´-GpYEEI and [DipR]5 was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin suggesting that the uptake for [DipR]5 may not be endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicates that [DipR]5 and Fluorescence-labeled [DipR]5 can act as a molecular transporter of several compounds including the cell-impermeable phosphopeptide and siRNA specifically.
The antibacterial activities of peptides were evaluated against pathogens. The minimum inhibitory concentrations (MIC) of peptides were determined by micro-broth dilution protocol against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. [DipR]5 and((DipR)2(WR)3) showed promising MIC values of 0.39-6.25 µM against Gram-positive bacteria strains and 3.13-25 µM against Gram-negative bacteria strains.
"]},{"key":"dc:source","label":"Dc Source","values":["Salehi, D. Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Tools and Antimicrobial Agents. [master’s thesis]. Irvine, CA: Chapman University; 2021. https://doi.org/10.36837/chapman.000221"]},{"key":"dc:title","label":"Title","values":["Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Tools and Antimicrobial Agents"]}]}],"canonical_facts":{"dc:contributor":["Dr. Keykavous Parang","Dr. Rakesh Kumar Tiwari","Dr. Simin Rahighi"],"dc:creator":["Salehi, David"],"dc:date.available":["2023-01-20T08:00:00Z"],"dc:description.abstract":["Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides.
The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F´-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]5 significantly improved the uptake of F´-GpYEEI, by approximately 110-folds after 3 h incubation. Confocal microscopy demonstrated the improved delivery of F´-GpYEEI (6-folds), which was mostly localized in the cytosol, in the presence of [DipR]5 for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F´-GpYEEI and [DipR]5 was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin suggesting that the uptake for [DipR]5 may not be endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicates that [DipR]5 and Fluorescence-labeled [DipR]5 can act as a molecular transporter of several compounds including the cell-impermeable phosphopeptide and siRNA specifically.
The antibacterial activities of peptides were evaluated against pathogens. The minimum inhibitory concentrations (MIC) of peptides were determined by micro-broth dilution protocol against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. [DipR]5 and((DipR)2(WR)3) showed promising MIC values of 0.39-6.25 µM against Gram-positive bacteria strains and 3.13-25 µM against Gram-negative bacteria strains.
"],"dc:identifier":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/15"],"dc:source":["Salehi, D. Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Tools and Antimicrobial Agents. [master’s thesis]. Irvine, CA: Chapman University; 2021. https://doi.org/10.36837/chapman.000221"],"dc:subject":["Cellular Uptake","Antimicrobial peptides","Nanoparticles","Diphenylalanine","Cancer and bacterial Infections","Gene Therapy","Amino Acids, Peptides, and Proteins","Bacterial Infections and Mycoses","Cancer Biology","Chemicals and Drugs","Medicinal and Pharmaceutical Chemistry","Medicinal Chemistry and Pharmaceutics","Nanomedicine","Nanotechnology","Nucleic Acids, Nucleotides, and Nucleosides","Other Pharmacology, Toxicology and Environmental Health","Other Pharmacy and Pharmaceutical Sciences","Pharmaceutical Preparations","Pharmaceutics and Drug Design","Pharmacology","Pharmacology, Toxicology and Environmental Health","Pharmacy and Pharmaceutical Sciences","Toxicology"],"dc:title":["Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Tools and Antimicrobial Agents"],"thesis:degree_discipline":["Pharmaceutical Sciences"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science (MS)"]},"updated_at":"2026-07-24T01:38:16Z"}