Date of Award
Spring 5-2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Pharmaceutical Sciences
First Advisor
Dr. Keykavous Parang
Second Advisor
Dr. Rakesh Tiwari
Third Advisor
Dr. Sun Yang
Fourth Advisor
Dr. Simin Rahighi
Abstract
Amphiphilic cyclic cell-penetrating peptides composed of an increasing number of alternative tryptophan (W) and arginine (R) were synthesized and evaluated as a molecular transporter and for their ability to deliver doxorubicin (Dox) and large molecular weight molecules, such as siRNA and proteins. We prepared a cyclic peptide containing alternative tryptophan (W) and arginine (R) residues and a lysine containing a free side chain amino group. Cyclic peptide [(WR)8WK βA] was conjugated through the free side chain amino group of β-alanine with Dox via a glutarate linker to afford [(WR)8WK]bA-Dox conjugate. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and triple-negative breast cancer cells, MDA-MB-231 and MCF-7, by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. Furthermore, [(WR)8WKbA]-Dox conjugate (5 μM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. The intracellular release of free Dox was assessed in CCRF-CEM cell line. The experiment exhibited that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h.Cyclic peptide [WR]9 efficiently increased the intracellular delivery of siRNA to triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) by 15-folds and 9-folds, respectively, compared to siRNA alone. Flow cytometry (FACS) and confocal microscopy confirmed the uptake of Alexa-Fluor siRNA (AF-488 siRNA) in the presence of [WR]9 at different concentrations. The calculated binding affinity (BC50) of siRNA to the peptide was 1.9, indicating strong binding for siRNA to the peptide. As a result, the peptide siRNA combination displayed only minimal silencing efficiency for signal transducer and activator of transcription 3 (STAT3). Furthermore, the peptide showed concentration and time-dependent cargo uptake when physically mixed with green fluorescent protein (GFP) and red fluorescent protein (RFP)as model proteins. [WR]9 was also able to internalize therapeutically relevant histone protein at different ratios.
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Recommended Citation
Zoghebi, K. Amphiphilic Cyclic Cell-Penetrating Peptides Containing Tryptophan and Arginine as Anticancer Agents and Drug Delivery System. [dissertation]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000344