Serum stability and physicochemical characterization of a novel amphipathic peptide C6M1 for siRNA delivery.

The efficient delivery of nucleic acids as therapeutic agents is a major challenge in gene therapy. Peptides have recently emerged as a novel carrier for delivery of drugs and genes. C6M1 is a designed amphipathic peptide with the ability to form stable complexes with short interfering RNA (siRNA). The peptide showed a combination of random coil and helical structure in water but mainly adopted a helical conformation in the presence of anions or siRNA. Revealed by dynamic light scattering (DLS) and microscopy techniques, the interaction of C6M1 and siRNA in water and HEPES led to complexes of ∼70 and ∼155 nm in size, respectively, but showed aggregates as large as ∼500 nm in PBS. The time-dependent aggregation of the complex in PBS was studied by DLS and fluorescence spectroscopy. At molar ratio of 15∶1, C6M1 was able to completely encapsulate siRNA; however, higher molar ratios were required to obtain stable complexes. Naked siRNA was completely degraded in 4 h in the solution of 50% serum; however C6M1 protected siRNA against serum RNase over the period of 24 h. Western blotting experiment showed ∼72% decrease in GAPDH protein level of the cells treated with C6M1-siRNA complexes while no significant knockdown was observed for the cells treated with naked siRNA.

Modification of a designed amphipathic cell-penetrating peptide and its effect on solubility, secondary structure, and uptake efficiency.

The development of safe and efficient nonviral gene delivery carriers has received a great deal of attention in the past decade. A class of amphipathic peptides has shown to be able to cross cell membranes and deliver cargo to the intracellular environment. Here, we introduce an 18-mer amphipathic peptide, C6M1, as a modified version of peptide C6 for short interfering RNA (siRNA) delivery. The importance of tryptophan residues and the effect of peptide sequence modification on its solubility, secondary structure, cytotoxicity, and uptake efficiency were investigated. The solubility of C6M1 in aqueous solutions was greatly enhanced compared to that of C6, confirmed by surface tension and anilinonaphthalene-8-sulfonic acid fluorescence measurements. C6M1 had a random/helical structure in water with the ability to attain a helical conformation in the presence of anionic components or membrane-mimicking environments. The modification significantly reduced the cytotoxicity of the peptide, making it a safer carrier for siRNA delivery. C6M1 was also found ∼90% more efficient than C6 in delivering Cy3-labeled siRNA in Chinese hamster ovary cells.