Date of Award
Doctor of Philosophy (PhD)
Rakesh K. Tiwari
Hamidreza Montazeri Aliabadi
Post-transcriptional gene silencing (PTGS), also termed as RNA interference (RNAi) possess a great therapeutic potential. Three siRNA-based products have been recently approved that have developed a renewed interest in the scientific community to explore this alternative therapeutic area rigorously. However, several challenges are limiting their use as a widespread therapeutic agent. The limitations include their membrane impermeability, short half-life, elimination by the kidney, entrapment by the reticuloendothelial system, sequestration by plasma proteins, endosomal trap, and off-target effects.
Several delivery systems and transfection agents have been investigated to address these challenges. Still, most of the systems suffer from toxicity and low efficiency. Hence, there is a critical need for developing non-toxic and more efficient siRNA delivery tools.
For the last two decades, it has been found that cell-penetrating peptide (CPP)-based delivery systems show enhanced uptake of siRNA. This dissertation aimed to investigate fatty acyl cell-penetrating peptides (CPPs) as siRNA delivery tools. We hypothesized that conjugation of hydrophobic molecules to the (HR)n peptide will enhance hydrophobicity, modulate cytotoxicity, and provide an efficient delivery system for siRNA. Therefore, we synthesized peptide conjugates with the sequence X-(R)n-(HR)4, and X-RnH4 (where X was a flexible hydrophobic chain (oleic acid) or a conformationally constrained steroidal hydrophobic molecule (cholesterol) and n = 0, 3, 4 or 5). Varying the arginine residues at the N-terminal helped us explore the structure-activity relationship of the peptide conjugate. Also, we synthesized another series of peptides with the sequence X-(HR)4-CGKRK, where X was either XI cholesterol, oleic acid or polyethylene glycol (PEG). Among the studied peptides, Olyel-R4-(HR)4, Oleyl-R5-(HR)4, Oleyl-K-(HR)4-CGKRK, and Cholesteryl-K-(HR)4-CGKRK demonstrated convincing data for siRNA delivery in MDA-MB-231 cells. However, X-RnH4 peptides were cytotoxic to all the studied cell lines, such as MDA-MB-231, MCF-7 and MCF-10A, even at the 5 μM range. We tested the cellular uptake of some of the conjugates for siRNA uptake, but these did not show significant uptake, so we stopped pursuing this series of peptides for further studies.
Oleyl conjugated histidine-arginine peptides with variable arginine residues proved to be non-cytotoxic at N/P 40 ( ~20 μM) in MCF-7, MCF-10A, and MDA-MB231 cells. Oleyl conjugates formed nanocomplexes with siRNA, possibly due to the presence of fatty acyl chain. Also, an increasing number of arginine residues in oleyl conjugated (HR)4 increased cellular uptake with subsequent silencing efficiency of the siRNA. Visual confocal images further strengthened the quantitative cellular internalization data obtained from flow cytometry. Oleyl-R5-(HR)4 resulted in ~80% inhibition of STAT-3 protein comparable to the commercially available transfection agent Lipofectamine 2000.
Furthermore, X-(HR)4-CGKRK conjugates were successfully synthesized and purified that formed nanocomplexes with siRNA with ~ diameter of/P ≥40. Cholesteryl-(HR)4-CGKRK caused even more silencing of STAT-3 in MDA-MB-231 (~ 89%) than oleyl-(HR)4-CGKRK (~82%) and lipofectamine (82%). Pegylated derivative of (HR)4-CGKRK caused ~60% inhibition of STAT-3. Further in-vivo exploration of cholesteryl and oleyl-CPPs may provide promising results in siRNA delivery.
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Sajid, MI. Synthesis, Characterization, and Evaluation of Amphiphilic Cell-Penetrating Peptides as siRNA Delivery Tools for Cancer Therapy. [dissertation]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000366
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