Investigating Hybrid Cyclic/Linear and Linear Peptides as Vehicles for Nucleic Acid Delivery
Small interfering RNA (siRNA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated Protein 9 (Cas9) are advanced biotechnological approaches that have been able to temporarily downregulate gene expression through post-transcription RNA interference or permanently knockdown a protein via creating a mismatch in DNA, respectively. Nucleic acids are anionic and hydrophilic molecule, which cannot enter the cell through passive diffusion and/or interaction with the cell membrane. Cell-penetrating peptides (CPPs) offer a unique carrier for nucleic acid delivery, where positively charged components of CPP bind to the negatively charged nucleic acid and the hydrophobic components enhance the interaction with cell membrane, and therefore, deliver nucleic acids to targeted cells. Arginine/tryptophan-containing peptides provide both characteristics and have been shown to be efficient in delivering nucleic acids. In this project, we evaluated a library of novel linear ((WR)XK(WR)5; X = 1-5) and cyclic ([(RW)5K](RW)X; X = 1-5) peptides for their physical characteristics, interaction with nucleic acids, cytotoxicity, internalization into cancer cells, and for their ability to promote and enhance the delivery of nucleic acids. Our data showed that these specific peptide structures showed minimum toxicity to the selected cell lines used in this project. These carriers also demonstrated strong binding affinity to siRNA, and therefore, despite effective cellular internalization of both siRNA and CRISPR/Cas9, did not show expected silencing/transfection efficiency, which could be due to poor intracellular release of delivered nucleic acids. Addition of an extra negatively charged component to the peptide/siRNA complexes enhanced the silencing efficiency. These data suggest that optimization of the complexes formed with these peptides could potentially provide a superior peptide-based nucleic acid carrier. Future in vitro and in vivo experiments are required to future investigate the efficacy of these carriers.
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