Characterization and evaluation of a peptide-based siRNA delivery system in vitro

Abstract

Since its inception more than a decade ago, gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases. However, the use of siRNA is hampered by its rapid degradation and poor cellular uptake in vitro and in vivo. Recently, peptide-based carriers have been applied to siRNA delivery, as an alternative to the traditional delivery systems. Here, a histidine-containing amphipathic amino acid pairing peptide, C6M3, which can form complexes with siRNA, was used as a new siRNA delivery system. This peptide exhibited a high affinity for siRNA and ability to efficiently deliver siRNA into the cells. The interaction of C6M3 with siRNA was investigated to determine the loading capacity of C6M3 at different peptide/siRNA molar ratios. At C6M3/siRNA molar ratio of 10/1, siRNA molecules were entirely associated with C6M3 as indicated by a gel electrophoretic assay and further confirmed by zeta potential analysis. The particle size distribution of the C6M3-siRNA complexes was studied using dynamic light scattering, which showed an intensity-based size distribution peaked approximately at 100 nm in RNase-free water and 220 nm in the Opti-MEM medium. C6M3 adopted a helical secondary structure in RNase-free water and became more so after forming complexes with siRNA. The interaction of siRNA with C6M3 is an entropy-driven spontaneous process, as determined by isothermal titration calorimetry (ITC) study. The efficiency of cellular uptake of the siRNA complexes at different C6M3/siRNA molar ratios was evaluated, and the results showed that C6M3 promoted efficient cellular uptake of siRNA into cells. Furthermore, a significant level of GAPDH gene silencing efficiency (69%) was achieved in CHO-K1 cells, with minimal cytotoxicity.

Peptide modified polycations with pH triggered lytic activity for efficient gene delivery

“…However, obvious hemolysis was detected when the pH of the culture media was 5.7, and the lytic activity showed concentration dependence. Previous work demonstrated that the EC 50 (the concentration of free peptide for 50% hemolysis) value of C6M3 was ∼16 μg mL −1 , 52 which was much lower than those in our work (29.7, 27.0 and 33.8 μg mL −1 for PDMAEMA-C6M3, PEI-C6M3 and PLL-C6M3, respectively). This may be attributed to the fact that the conjugation of C6M3 to polycations compromises the lytic activity.…”

“…51 Previous studies showed that the C6M3 peptide (a peptide derived from C6) features pH-triggered lytic activity, and promising membrane lysis was observed under endo/lysosomal acidic conditions rather than at a normal pH value. 51,52 We hypothesize that the incorporation of polycations with C6M3 could not only enhance the gene delivery efficiency through the promotion of endo/lysosomal escape but also avoid off-site toxicity. As shown in Scheme 1, the C6M3 modified polycations condense DNA into nanoparticles, which can be endocytosed by cells through the endosome pathway.…”

Dual role of the miR‐146 family in rhinovirus‐induced airway inflammation and allergic asthma exacerbation

“…Generally, the therapeutic application of miRNA mimics is limited due to their low capacity to penetrate cell membranes due to their negative charge. 70 To date, many substances have been assessed for the delivery of nucleic acids, including CPPs, but there is still a lack of efficient, safe, and specific methods for the in vivo delivery of potentially therapeutic nucleic acids, including miRNA mimics. 71,72 We previously demonstrated the capacity of CPP-miR-146a noncovalent nanocomplexes to suppress inflammatory responses in cell cultures and a mouse model of irritant contact dermatitis.…”

DNA- and RNA-binding ability of oligoDapT, a nucleobase-decorated peptide, for biomedical applications

“…The importance of new drugs able to bind doublestranded DNA, as well as to interfere with double-stranded RNA-driven biological processes [39][40][41] or act as stabilizers and carriers of double-stranded RNA drugs, [42][43][44][45][46] prompted us to study the interaction of oligoDapT with dA 12 /dT 12 and poly(rA)/poly(rU), chosen as double-stranded DNA and RNA model systems, respectively. First, the target complexes were prepared by mixing equimolar amounts of DNA and RNA single strands, heating the resulting solutions at 90°C for 10 minutes and then slowly cooling overnight.…”