Design and characterization of a new peptide vector for short interfering RNA delivery

Abstract

RNA interference holds tremendous potential as one of the most powerful therapeutic strategies. However, the properties of short interfering RNA (siRNA), such as hydrophilicity, negative charge, and instability in serum have limited its applications; therefore, significant efforts have been undertaken to improve its cellular uptake. Cell penetrating peptides have been utilized to deliver various biologically active molecules, such as proteins, liposomes, nanoparticles, peptide nucleic acids, and recently small interfering RNAs. Here, we introduce a new cell penetrating peptide GL1(Ac-GLWRAWLWKAFLASNWRRLLRLLR-NH2) to improve the intracellular uptake of siRNA. This peptide consists of four tryptophan residues that facilitated its binding with the cell membrane, five arginine residues and one lysine residue which are positively charged at physiological pH, which induced the formation of peptide-siRNA complexes and enhanced the affinity of the peptide and cell membrane. Moreover, GL1 adopted helical secondary structure due to the altered distribution of polar and nonpolar residues in the sequence. In this study, we investigated the effect of peptide/siRNA molar ratio on the particle size, surface charge, secondary structure, and uptake efficiency. The results showed that GL1 formed stable complexes with siRNA mainly through electrostatic interaction and hydrophobic interaction, and the complexes displayed a spherical shape with the size of ~100 nm and positive surface charge. Utilizing the techniques of fluorescence microscopy and flow cytometry, the intracellular localization of Cy3-labeled GAPDH siRNA was visualized and the cellular uptake was quantified. It is worth noting that in the serum free environment, compared to Lipofectamine 2000, GL1 achieved higher cellular uptake of siRNA (~95%); in the presence of serum, GL1 retained the same level of siRNA cellular uptake (~84%) as Lipofectamine 2000. In addition, the viability of cells treated by GL1 in all studied molar ratios was >85%, which was significantly higher than that treated by Lipofectamine 2000 (~70%). Taken together, the peptide GL1 demonstrated promise as a siRNA delivery system.

Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

“…These factors including positive surface charge and small particle size must have contributed to the increased transfection efficiency. Our results were supported by other studies indicating that the amount of positive charges on the cell surface and the size of the vector/oligonucleotide complex played important roles in determining successful gene delivery [41–43]. A positively charged structure is desirable since it can preferentially adhere to the negatively charged cell surface receptors, leading to endocytosis.…”

CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides

“…Cell-penetrating peptides (CPPs) are short peptides (<30 amino acids), often cationic and have the ability to internalize into the eukaryotic cells without causing significant membrane damage ( 1 – 2 ). Owing to this intrinsic cell-penetrating property, CPPs are capable of delivering conjugated therapeutic molecules like proteins ( 3 ), peptides ( 4 ), nucleic acid ( 5 – 8 ), small molecule drugs ( 9 ), nanoparticles ( 10 – 11 ), etc. to the cells and tissues.…”

The Formation of Nanoparticles between Small Interfering RNA and Amphipathic Cell-Penetrating Peptides

“…For example, CPP CADY with +5 charge and no fatty acid modification has estimated siRNA binding to CPP K D at 15 nmol/L concentrations in MQ 37, 38. Another study reported a K D  value of 224 nM in MQ measured by iTC for a peptide with charge 5.0–5.5 in water 25 . For a C6 peptide with seven arginines in its sequence, a K value of 9.23 × 10 6 M −1 was reported in water, resulting in a K D  value of ∼108 nM 26 .…”

“…This change of net charge affects the interactions between a CPP and the cargo. Isothermal calorimetry (iTC) is an accurate method that has been used to study mainly protein-ligand interactions, but a growing number of studies harness iTC to study CPP-ligand interactions, 24 CPP-siRNA interactions,25, 26 and CPP interactions with membranes 27 . Furthermore, iTC enables to determine the binding constant of a CPP to cargo and calculate values of apparent dissociation constants.…”