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 190 nm in diameter. p5RHH exhibits high efficiency with GFP knockdown at concentrations as low as 5 nM, 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.

Figure 1

A Optimization of p5RHH/siRNA ratios reveal an increasing transfection efficiency with increasing amounts of p5RHH until a maximum at 150:1 p5RHH:siRNA. B Alamar blue assays indicate no cytotoxicity at p5RHH:siRNA ratios up to 200:1 when transfecting 50nM siRNA.

Figure 2

A Gel retardation assays show that a p5RHH:siRNA ratio of 50:1 is required to completely complex siRNA. B Analysis of p5RHH:siRNA nanoparticles by dynamic light scattering and zeta potential analysis suggest that effective surface charge determines particle size. Nanoparticles with a surface charge of larger magnitude exhibit smaller diameters suggesting the importance of electrostatic interactions in stabilizing p5RHH:siRNA nanoparticles. A p5RHH:siRNA ratio of 100:1 generates the smallest particle size of 190nm. C SEM analysis of particle size confirms the dynamic light scattering data revealing small complexes of 100–150nm in diameter. (scale bar 100nm)

Figure 3

A Dose response by flow cytometry shows that p5RHH mediated transfection is less efficient than Lipofectamine2000, but also shows high siRNA transfection efficiency with visible knockdown at concentrations as low as 5nM. B Alamar blue assays indicate that p5RHH exhibits minimal toxicity at siRNA concentrations up to 200nM. Western blotting (C) and RT-PCR (D) analysis of GFP mRNA confirms the ability of p5RHH to decrease mRNA levels in a sequence specific manner with an IC50 of ~50nM. Lipofectamine 2000 has a higher transfection efficiency with an IC50 of between 10–25nM as determined by western blotting (E) and RT-PCR (F).

Figure 4

A The nonfunctioning melittin derivative, p5RWR, exhibits no siRNA transfection ability when screened for knockdown of GFP in B16GFP cells via flow cytometry. siRNA is being delivered, but does not reach cytoplasm until incubated with 50µM chloroquine. Confocal microscopy confirms the flow cytometry data, indicating that p5RWR(B) does not manifest appreciable oligonucleotide release into the cytoplasm unless incubated with 50µM chloroquine (C). D In comparison, p5RHH shows efficient oligo release into the cytoplasm similar to oligonucleotide delivery via Lipofectamine 2000.

Figure 5

Western blotting data indicate that p5RHH (A) is approximately 5-fold less efficient than Lipofectamine 2000 (B) at initiating a decrease in STAT3 protein levels in B16 cells. RT-PCR data show that p5RHH (C) loses activity at concentrations below 50nM while Lipofectamine 2000 (D) exhibits activity at doses as low as 10nM. B16 viability analysis via Alamar Blue demonstrates that p5RHH (E) transfection leads to a decrease in B16 viability by silencing oncogene expression in a sequence specific manner whereas Lipofectamine2000 (F) causes nonspecific dose dependent cytotoxicity.

Figure 6

A Western blotting depicts a dose dependent decrease in STAT3 protein levels in HUVECs treated with STAT3 specific siRNA. B RT-PCR data illustrate a p5RHH-dependent 60% knockdown in STAT3 mRNA at concentrations as high as 200nM. C p5RHH has no cytotoxicity towards HUVEC cells when transfecting siRNA. D HUVECs treated with STAT3 siRNA show a 60% decrease in tube formation on matrigel when compared to controls (E, F) as quantified in G. A decrease in tube formation is accompanied by a 40% decrease in HUVEC migration in response to bFGF in transwell migration assays as determined by microscopy (H) and Alamar Blue assays (I)

Figure 7

A Western blot analysis illustrates knockdown of JNK2 in RAW264.7 cells by p5RHH with IC50 <25nM. B Importantly, p5RHH causes only a ~5% decrease in cell viability when transfecting scrambled siRNA at 100nM. C–E Knockdown of JNK2 at 50nM siRNA shows a strong decrease in lipid droplet accumulation in RAW264.7 cells when incubated with 50µg/mL Ac-LDL overnight when compared to cells treated with Scrambled siRNA and untreated controls.

Figure 8

A Incubation of p5RHH:siRNA nanoparticles with 500µg/mL HSA for 30 minutes or overnight are characterized by improved GFP knockdown when compared to freshly prepared p5RHH:siRNA nanoparticles. B Particle size analysis of p5RHH:siRNA incubated in the presence of serum albumin overnight reveal a stable size. C Confocal microscopy of B16 cells transfected in normal cell culture media supplemented with 10% FBS shows efficient oligonucleotide release into the cytoplasm.