Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery

Abstract

RNA interference (RNAi) is a powerful tool capable of targeting virtually any protein without time-consuming and expensive drug development studies. However, due to obstacles facing efficient and safe delivery, RNAi-based therapeutic approach remains a challenge. Herein, we have designed and synthesized a number of disulfide-constraining cyclic and hybrid peptides using tryptophan and arginine residues. Our hypothesis was that peptide structures would undergo reduction by intracellular glutathione (more abundant in cancer cells) and unpack the small interfering RNA (siRNA) from the peptide/siRNA complexes. A subset of newly developed peptides (specifically, C4 and H4) exhibited effective cellular internalization of siRNA (∼70% of the cell population; monitored by flow cytometry and confocal microscopy), the capability of protecting siRNA against early degradation by nucleases (monitored by gel electrophoresis), minimal cytotoxicity in selected cell lines (studied by cell viability and LC50 calculations), and efficient protein silencing by 70–75% reduction in the expression of targeting signal transducer and activator of transcription 3 (STAT3) in human triple-negative breast cancer (TNBC) MDA-MB-231 cells, analyzed using the Western blot technique. Our results indicate the birth of a promising new family of siRNA delivery systems that are capable of safe and efficient delivery, even in the presence of nucleases.

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools

“…Our previous studies showed that cyclic CPPs, [WR] 4 and [WR] 5 , with alternation of tryptophan (W) and arginine (R) residues, increased the cellular uptake of different compounds such as phosphopeptides, doxorubicin, ,,, paclitaxel, camptothecin, curcumin, , anti-HIV drugs, , gadolinium nanoparticles, selenium nanoparticles, gold nanoparticles, , and oligonucleotides. , Cyclic CPPs containing alternate W and R residues demonstrated improved cellular uptake delivery when compared to linear CPPs . Moreover, the uptake of both [WR] 4 and [WR] 5 was not dependent on the endocytosis pathway. , …”

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines

“…Other chemical reagents and solvents were purchased from Millipore-Sigma (Milwaukee, WI, USA) and used without further purification. More details regarding the materials used in the present study have been previously described [ 7 ].…”

“…The cellular uptake experiment was conducted on the breast cancer cell line MDA-MB-231. The detailed protocol is provided in our recently published manuscript [ 7 ]. Briefly, cells were seeded in each well of the 24-well plate (approximately 200,000 cells per well) and were allowed to settle in the incubator at 37 °C for 24 h. The peptide and siRNA complexes were made at N to P ratios of 20 and 40, where the final concentration of siRNA in each complex was 36 nM, and were allowed to incubate for 30 min at room temperature.…”

“…Cell-penetrating peptides (CPPs) provide promising carriers for the intracellular delivery of nucleic acids [ 6 ]. CPPs are effective in delivering a wide range of therapeutic molecules, including small molecules, nucleic acids, and proteins with limited toxicity, and are currently further explored in drug delivery studies [ 6 , 7 , 8 , 9 ]. A net cationic charge is a common physicochemical property of most CPPs and is known to facilitate membrane internalization [ 10 ].…”

“…We have also reported siRNA delivery via a wide range of specifically designed peptides, including hybrid cyclic peptides (that incorporate arginine (R) residues in a ring and tryptophan (W) residues in a hydrophobic chain) and cyclic peptides with a disulfide bridge [ 7 , 8 ]. These sequences are designed to resolve the issue of toxicity and poor pharmacokinetic/pharmacodynamic profiles observed with polyethyleneimines (PEI) and poly-L-lysine.…”