We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and ?-polyglutamic acid (?-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 ?-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 ?-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of ?-PGA content, which decreased the ?-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including ?-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/?-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.
We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, ?-polyglutamic acid (?-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/?-PGA complexes) were shown to be stable nanoparticles, and those with ?-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/?-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/?-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/?-PGA complexes for clinical gene therapy.
The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with polyamidoamine (PAMAM) dendrimer and chondroitin sulfate (CS) for effective and secure gene delivery. PAMAM dendrimers are new cationic polymers that are expected to be used as gene delivery vectors. However, cationic non-viral gene vectors showed cytotoxicity by binding to negative cellular membranes. We therefore prepared a ternary complex by adding CS, an anionic polymer, and examined its usefulness. The pDNA/PAMAM dendrimer complex (PAMAM dendriplex) and the PAMAM dendriplex coated by CS (CS complex) showed nanoparticles with positive ?-potential and negative ?-potential, respectively. The CS complex had no cytotoxicity against B16-F10 cells and no agglutination activity, although severe cytotoxicity and high agglutination were observed in the PAMAM dendriplex. As a result of an in vitro gene expression study of B16-F10 cells, not only the PAMAM dendriplex but also the CS complex showed high transfection efficiency. The transfection efficiency of the CS complex was significantly inhibited by clathrin-mediated endocytosis inhibitor (chlorpromazine), caveolae-mediated endocytosis inhibitor (genistein), and hypothermia. Tail-vein injection of the CS complex into mice led to significantly higher gene expression in the spleen than the PAMAM dendriplex. Thus, the ternary complex of pDNA electrostatically assembled with PAMAM denriplex and CS showed safe high gene expression in the spleen. This vector is expected to be useful for useful gene delivery.
In this experiment, we developed a novel safe and effective gene delivery vector coated with ?-polyglutamic acid (?-PGA-coated complexes). The ?-PGA-coated complex was composed of chiseled spherical nano-particles with anionic charges. The plasmid DNA/polyethyleneimine complex (non-coated complex) showed high transgene efficiency in the spleen and lung after intravenous administration in mice, with high liver toxicity and lethality. On the other hand, ?-PGA-coated complex selectively showed high transgene efficiency in the spleen without such toxicity. Furthermore, the ?-PGA-coated complex highly accumulated and showed high gene expression in the marginal zone of the spleen. Those results strongly indicated that ?-PGA-coated complex was suitable as a DNA vaccine vector. We therefore applied ?-PGA-coated complex to melanoma DNA vaccine, pUb-M. The ?-PGA-coated complex containing pUb-M significantly inhibited the growth and metastasis of a melanoma cell line, B16-F10 cells. In conclusion, we developed a splenic gene vector, ?-PGA-coated complex, as a novel technology for clinical vaccination.
The purpose of the present study was to investigate the usefulness of the ternary complex with protamine and ?-polyglutamic acid (?-PGA), which are biodegradable materials for foods and medical products, as a safe gene delivery vector. We formed cationic binary complexes (plasmid DNA (pDNA)/protamine complexes) with high transfection efficiency. The binary complex showed slight toxicity probably related to its total cationic charge. Then, we formed ternary complexes (pDNA/protamine/?-PGA complexes) by addition of anionic polymer, ?-PGA, and they showed no cytotoxicity. The transfection efficiency of the pDNA/protamine/?-PGA complexes was as high as that of the pDNA/protamine complexes, although their zeta potentials were different. Inhibition study of the gene expressions in B16-F10 cells suggested that pDNA/protamine complexes were taken up by caveolae-mediated endocytosis and macropinocytosis. On the other hand, pDNA/protamine/?-PGA complexes were taken up by clathrin-mediated endocytosis and macropinocytosis. Thus, we succeeded in developing the ternary complex as a safe gene delivery vector with biocompatible materials.
We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3?-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of ?-polyglutamic acid (?-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by ?-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by ?-PGA did not show significant cytotoxicity. The ternary complexes coated by ?-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use.
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