Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Tools and Antimicrobial Agents

<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]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]₅and Fluorescence-labeled [DipR]₅ 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]₅and((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.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2021

Author dc:creator

• Salehi, David

Contributors dc:contributor

• Dr. Keykavous Parang
• Dr. Rakesh Kumar Tiwari
• Dr. Simin Rahighi

• 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

Repository record dc:identifier

https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/15

OAI identifier oai:identifier

oai:digitalcommons.chapman.edu:pharmaceutical_sciences_theses-1015