Melittin derived peptides for nanoparticle based siRNA transfection

Abstract

Traditional transfection agents including cationic lipids and polymers have high efficiency but cause cytotoxicity. While cell penetrating peptide based transfection agents exhibit improved cytotoxicity profiles, they do not have the efficiency of existing lipidic agents due to endosomal trapping. As a consequence, we propose an alternative method to efficient peptide based siRNA transfection by starting with melittin, a known pore-forming peptide. By incorporating modifications to decrease cytotoxicity and improve siRNA binding, we have developed p5RHH, which can complex siRNA to form nanoparticles of 190nm in diameter. p5RHH exhibits high efficiency with GFP knockdown at concentrations as low as 5nM, with negligible cytotoxicity. To date, p5RHH has shown the ability to transfect B16 cells, Human Umbilical Vein Endothelial Cells, and RAW264.7 cells with high efficiency. These in vitro models demonstrate that p5RHH mediated transfection can block cancer cell proliferation, angiogenesis, and foam cell formation. Moreover, p5RHH/siRNA nanoparticles maintain their size and transfection efficiency in the presence of serum proteins suggesting the potential for use of p5RHH in vivo. These data suggest that our strategy for development of siRNA transfecting peptides can provide an avenue to safe and effective siRNA therapeutics.

Peptide-siRNA nanocomplexes targeting NF-κB subunit p65 suppress nascent experimental arthritis

“…tions mitigate the undesirable pore-forming capacity of peptide, yet retain its ability to condense siRNA and facilitate endosomal escape, as previously reported (13,16). A simple mixing procedure of only 10 minutes yields a complex that is small enough (~55 nm) to passively diffuse rapidly into inflamed tissues, where it is retained, yet avoids hepatic sequestration (14).…”

“…Moreover, these agents tend to aggregate with serum proteins in vivo and often mediate robust complement activation with potential deleterious effects (34)(35)(36). We recently reported the development of an amphipathic peptide-based siRNA nanoplatform (13,14) that uses a modified version of the bee venom component melittin to transport siRNA (15). Melittin is a well-studied peptide with pore-forming features that derive from its ability to insert into lipid membranes stably through hydrophobic interactions among resident membrane phospholipids interacting with its α-helical peptide secondary structure (16,37,38).…”

“…A simple mixing procedure of only 10 minutes yields a complex that is small enough (~55 nm) to passively diffuse rapidly into inflamed tissues, where it is retained, yet avoids hepatic sequestration (14). Using a mouse model of antibody-mediated inflammatory arthritis, we found that anti-NF-κB peptide-siRNA self-assembling nanocomplexes (13,14) effectively silenced the expression of p65 as well as a broad array of downstream cytokine effectors in the inflamed paws, and also suppressed arthritis with minimal off-target molecular effects.…”

“…Accordingly, in the present work, we sought to define the utility of a novel siRNA delivery approach using nanoparticles targeted to the NF-κB signaling subunit p65, which is a principal transcriptional regulator of the canonical NF-κB pathway (12). The nanoparticle construct is composed of a self-assembling peptide-siRNA complex that serves to protect the siRNA from serum deactivation, while avoiding reticuloendothelial system uptake and delivering functional oligonucleotides to selected inflammatory targets (13,14). Key features of the complex that promote sequentially coordinated endosomal uptake, endosomal lysis, and siRNA release depend on specific molecular features of the carrier peptide (13), which is derived through modification of the bee venom peptide melittin (15).…”

“…The nanoparticle construct is composed of a self-assembling peptide-siRNA complex that serves to protect the siRNA from serum deactivation, while avoiding reticuloendothelial system uptake and delivering functional oligonucleotides to selected inflammatory targets (13,14). Key features of the complex that promote sequentially coordinated endosomal uptake, endosomal lysis, and siRNA release depend on specific molecular features of the carrier peptide (13), which is derived through modification of the bee venom peptide melittin (15). Selected amino acid truncations and substituThe NF-κB signaling pathway is implicated in various inflammatory diseases, including rheumatoid arthritis (RA); therefore, inhibition of this pathway has the potential to ameliorate an array of inflammatory diseases.…”