Date of Award
Winter 1-2021
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Pharmaceutical Sciences
First Advisor
Dr. Keykavous Parang
Second Advisor
Dr. Rakesh Kumar Tiwari
Third Advisor
Dr. Simin Rahighi
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.
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Recommended Citation
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
Included in
Amino Acids, Peptides, and Proteins Commons, Bacterial Infections and Mycoses Commons, Cancer Biology Commons, Medicinal and Pharmaceutical Chemistry Commons, Medicinal Chemistry and Pharmaceutics Commons, Nanomedicine Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Other Pharmacology, Toxicology and Environmental Health Commons, Other Pharmacy and Pharmaceutical Sciences Commons, Pharmaceutical Preparations Commons, Pharmaceutics and Drug Design Commons, Pharmacology Commons, Toxicology Commons
Comments
Contact Information : mssalehi@ucdavis.edu