Novel siRNA Delivery System Using a Ternary Polymer Complex with Strong Silencing Effect and No Cytotoxicity
Novel siRNA Delivery System Using a Ternary Polymer Complex with Strong Silencing Effect and No Cytotoxicity
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DOI：
10.1248/bpb.b14-00058
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发表时间：
2014-08-01
影响因子：
2
通讯作者：
Sasaki, Hitoshi
中科院分区：
文献类型：
--
作者：
Kodama, Yukinobu;Shiokawa, Yumi;Sasaki, Hitoshi
我们利用聚乙烯亚胺（PEI）和γ-聚谷氨酸（γ-PGA）的三元复合物开发了一种新型的小干扰RNA（siRNA）递送系统，该系统具有沉默效果且无细胞毒性。siRNA与PEI的二元复合物的粒径约为73- 102 nm，ζ电位约为45-52 mV。siRNA/PEI复合物的沉默效应随着PEI的增加而增加，并且具有大于16的电荷比的siRNA/PEI复合物在规律表达荧光素酶的小鼠结肠癌细胞系（Colon 26/Luc细胞）中显示出显著的荧光素酶敲低。然而，在siRNA/Lipofectamine复合物和siRNA/PEI 16复合物中观察到强细胞毒性和血液凝集。据报道，用阴离子化合物再充电阳离子络合物是克服这些毒性的一种有前途的方法。因此，我们通过添加γ-PGA来制备siRNA与PEI（电荷比16）的三元复合物以降低细胞毒性并递送siRNA。正如预期的那样，三元复合物的细胞毒性随着γ-PGA含量的增加而降低，这降低了复合物的c-电位。在包含具有阴离子表面电荷的γ-PGA的三元复合物中发现了与siRNA/Lipofectamine复合物相当的强沉默作用。用荧光显微镜证实三元复合物高度掺入Colon 26/Luc细胞中。在实现外源转染基因的敲低后，在B16-F10细胞中评估复合物介导内源管家基因甘油醛3-磷酸脱氢酶（GAPDH）敲低的能力。三元复合物（siRNA/PEI 16/γ-PGA 12复合物）表现出显著的GAPDH敲低效应。因此，我们开发了一种有用的siRNA递送系统。
We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and gamma-polyglutamic acid (gamma-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102nm in particle size and 45-52 mV in zeta-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 gamma-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of gamma-PGA content, which decreased the c-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including gamma-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/gamma-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.