Serum Stability and Physicochemical Characterization of a Novel Amphipathic Peptide C6M1 for SiRNA Delivery

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.

Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

“…This determined also the behavior of these polyplexes in the gel retardation assay. These data are in agreement with those obtained in other similar studies with polycations (Jafari et al, 2014). Based on these results, we chose to carry on the following experiments with two ratios, one corresponding to a condition of partially complexation (w/w of 0.5 = R1) and another corresponding to a full complexation (w/w of 5 = R2).…”

Development of novel self-assembled polymeric micelles from partially hydrolysed poly(2-ethyl-2-oxazoline)-co-PEI-b-PCL block copolymer as non-viral vectors for plasmid DNA in vitro transfection

“…Hence, all micelle/pDNA complexes were prepared in HEPES buffer due to the low ionic strength of buffer for further experiments. Also our results compatible with the Jafari et al [25] indicated that the media were considerably important on the particle size and charge. Moreover, these results indicated that both PPP30 and PPP60 micelle/DNA complexes had desirable size for biological applications and could be passively targeted by EPR effect [26,27].…”

“…The results exhibited that both PPP30 and PPP60 micelle/DNA complexes had excess positive charge at highest N/P ratio (Figure 3(B,F)). These results supported the observations of particle size that excess cationic charge leading to small particle size due to high repulsion of particles causes to prevent the aggregation [9,25]. Hence, all micelle/pDNA complexes were prepared in HEPES buffer due to the low ionic strength of buffer for further experiments.…”

Delivery of therapeutic oligonucleotides with cell penetrating peptides

“…Similar approaches were used for other non-covalent CPPs such as Pep-3 for HypNA-pPNA [49], CADY, C6 and MPEG-PCL-CH2R4H2C for siRNA condensation [126][127][128][129]. Fluorescence assays were also combined to circular dichroism (CD) analyses to monitor conformational changes that might occur in the presence of the ON and during CPP:ON complex formation [125,[128][129][130]. Although both fluorescence and CD investigations suggest interactions and formation of CPP:ON complexes, the use of gel shift assays has been generalized to demonstrate the formation of complexes [124,131].…”

“…For other CPP:ON complexes, the size and the biological activity clearly vary with the MR [50]. Size and charge can also depend on the environment which is important since nanoparticles stability and homogeneity might vary according to the nature of the buffer, as described for C6M1:siRNA particles [130]. In addition physiological conditions such as serum addition can influence colloidal properties.…”