Abstract
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.
Highlights
Over the past two decades, major advances have been made in the field of gene therapy
Peptide-short interfering RNA (siRNA) complexes were formed by adding peptide solution into siRNA in proportion according to the designed experiment and diluting in RNase free water, HEPES (6 mM, pH = 7.4), or phosphate buffered saline, Phosphate buffered saline (PBS), to achieve the final concentrations
Understanding the properties of peptides is necessary for their effective use as siRNA delivery systems
Summary
Over the past two decades, major advances have been made in the field of gene therapy. RNA interference (RNAi) has provided new perspectives in developing novel nucleic acid (NA)-based therapeutics [1,2,3]. Their development has been restricted by their poor stability and cellular uptake. For the clinical advancement of RNAi, the design and development of safe and effective delivery systems is vital. Several viral and non-viral delivery systems, including lipids [4,5], polymers [6,7], and peptides [8,9] have been engineered and developed to obtain desired capabilities to overcome the cellular delivery barriers
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