Novel siRNA Delivery System Using a Ternary Polymer Complex with Strong Silencing Effect and No Cytotoxicity

Abstract

We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/ γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system. Key words gene delivery; small interfering RNA; γ-polyglutamic acid; ternary complex Research into RNA interference (RNAi) has advanced rapidly since it was first discovered in 1998. 1) RNAi is a natural mechanism conserved in plant and mammalian cells that results in specific silencing of a target gene by short sequences of RNA, known as small interfering RNA (siRNA).2) siRNA is a double-stranded RNA (dsRNA) composed of 21-23 nucleotides, and its ability to induce sequence-specific RNAimediated down-regulation of complementary mRNA has been demonstrated, resulting in knockdown of a target gene protein at the post-transcriptional level. [3][4][5] Numerous studies have used siRNAs as potential therapeutic agents for treating various diseases, including cancer and other diseases as a result of genetic disorders or viral infection. [6][7][8] Despite the immense therapeutic potential of this technology, effective systemic and intracellular delivery of siRNA is quite limited because of its rapid degradation by plasma nucleases and poor penetration of the plasma membrane of target cells.9,10) Therefore, the establishment of an siRNA delivery system that enables prolonged activity without degradation by nucleases and efficient cytosolic delivery of siRNA into target cells is indisp...

Progress in the development of Lipoplex and Polyplex modified with Anionic Polymer for efficient Gene Delivery

“…Moreover, a ternary complex of pDNA/protamine/γPGA ( Figure 5A) was taken up by clathrinmediated endocytosis and macropinosytosis and showed high transfection ef iciency in B16-F10 cells [75]. In addition, a ternary complex of siRNA/PEI/γPGA ( Figure 5A) showed a high gene silencing effect and no cytotoxicity in B16-F10 cells [76]. From these indings, ternary complexes with γPGA may be a potential carrier for targeting tumor cells.…”

“…Anionic polymer or its conjugate Cationic polymer or liposome Reference pDNA ᵞPGA Chitosan [66,67] pDNA ᵞPGA Dendrigraft poly-L-lysine [80] pDNA ᵞPGA PEI [81][82][83] pDNA ᵞPGA Protamine [75] pDNA αPGA DOTAP/Chol liposome [54] siRNA ᵞPGA Chitosan [73] siRNA ᵞPGA Dendrigraft poly-L-lysine [74] siRNA αPGA DOTAP/Chol liposome [77] siRNA αPGA DMAPAP/DOPE liposome [78,79] siRNA ᵞPGA PEI [76] pDNA Heparin Cationic glycopolymer (Tr4) [84] pDNA Heparin-PEI - [85] pDNA Alginic acid, Pectin Lipofectamine 2000 [24] pDNA: plasmid DNA, siRNA: small interfering RNA, ᵞPGA: poly-ᵞ-glutamic acid, αPGA: poly-α-glutamic acid, PEI: polyethylenimine, DMAPAP: 2-{3-[bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methylacetamide, Chol: cholesterol, DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.…”

Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery

“…In this study, a high amount of PEI was used to prepare nanoparticles which showed high transfection efficiency ( Figure 3 B,C) but also exhibited severe cytotoxicity ( Figure 4 A,B). One promising approach to reduce the cytotoxicity is to incorporate or recharge anionic polymers into the cationic PEI complexes [ 26 , 29 , 30 , 53 ]. Therefore, in this study, γ-PGA was incorporated into γ-DDNPs and resulted in the high gene/siRNA transfection efficiency with minor cytotoxicity ( Figure 6 and Figure 7 ).…”

siRNA intervention inhibiting viral replication and delivery strategies for treating herpes simplex viral infection

“…However, during modifications, care should be elicited not to induce any changes in the siRNA specificity or their silencing activity. A novel siRNA delivery system was created with ternary complex of polyethyleneimine (PEI) and c-polyglutamic acid [14]. This complex displayed a prominent silencing activity almost equal to the lipofectamine complex with siRNA and no cytotoxicity being reported.…”