黄 伟 吕 明 高钟镐 金明姬 杨长青. mPEG-PCL-g-PEI聚合物纳米粒介导的小干扰RNA递送J. 药学学报, 2011,46(3): 344-349.
引用本文: 黄 伟 吕 明 高钟镐 金明姬 杨长青. mPEG-PCL-g-PEI聚合物纳米粒介导的小干扰RNA递送J. 药学学报, 2011,46(3): 344-349.
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HUANG Wei, Lv- Meng, Gao-Zhong-Gao, Jin-Meng-Ji, Yang-Chang-Jing. Small interfering RNA delivery mediated by mPEG-PCL-g-PEI polymer nanoparticlesJ. 药学学报, 2011,46(3): 344-349.
Citation: HUANG Wei, Lv- Meng, Gao-Zhong-Gao, Jin-Meng-Ji, Yang-Chang-Jing. Small interfering RNA delivery mediated by mPEG-PCL-g-PEI polymer nanoparticlesJ. 药学学报, 2011,46(3): 344-349.
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mPEG-PCL-g-PEI聚合物纳米粒介导的小干扰RNA递送

Small interfering RNA delivery mediated by mPEG-PCL-g-PEI polymer nanoparticles

    摘要
  • 摘要:

    本文旨在合成小干扰核糖核酸 (siRNA) 递送载体聚合物聚乙二醇-聚己内酯-聚乙烯亚胺 (mPEG-PCL-g-PEI), 并探讨其体外siRNA递送性能。通过开环聚合反应制备二嵌段共聚物聚乙二醇-聚己内酯 (mPEG-PCL-OH), 将mPEG-PCL-OH的羟基末端依次化学转化为羧基 (-COOH) 和N-羟基琥珀酰亚胺 (-NHS) 生成mPEG-PCL-NHS, 再将mPEG-PCL-NHS同枝化聚乙烯亚胺(PEI)反应生成三元共聚物mPEG-PCL-g-PEI。应用傅里叶变换红外光谱 (FTIR)、核磁共振 (NMR) 和凝胶渗透色谱 (GPC) 对聚合物mPEG-PCL-g-PEI进行结构表征; 通过复凝聚法制备mPEG-PCL-g-PEI/siRNA纳米粒, 并测定其粒径和zeta电位; 通过体外细胞MTT测试, 比较mPEG-PCL-g-PEI/siRNA纳米粒和PEI/siRNA纳米粒的细胞毒性; 通过体外细胞转染实验, 考察不同N/P比的mPEG-PCL-g-PEI/siRNA纳米粒对萤火虫荧光素酶基因表达的抑制效率。结果表明, 合成的三元聚合物 (mPEG5k-PCL1.2k)1.4-g-PEI10k能压缩siRNA形成粒径为50～200 nm的纳米粒, 其表面带有正电荷。MTT分析结果显示 (mPEG5k-PCL1.2k)1.4-g-PEI10k/siRNA纳米粒的细胞毒性显著小于PEI10K/siRNA纳米粒 (P < 0.05)。当N/P比在50～150, 萤火虫荧光素酶基因的表达显著下调 (P < 0.01)。当N/P比为125时, 萤火虫荧光素酶基因表达的抑制效率最大。聚合物mPEG-PCL-g-PEI能递送siRNA进入细胞, 抑制靶基因表达, 且细胞毒性较低, 有望成为一种新型的siRNA递送载体。

     

    Abstract:

    The aim of this paper is to report the synthesis of the mPEG-PCL-g-PEI copolymers as small interfering RNA (siRNA) delivery vector, and exploration of the siRNA delivery potential of mPEG-PCL-g-PEI in vitro.  The diblock copolymers mPEG-PCL-OH was prepared through the ring-opening polymerization.  Then, the hydroxyl terminal (-OH) of mPEG-PCL-OH was chemically converted into the carboxy (-COOH) and N-hydroxysuccinimide (NHS) in turn to prepare mPEG-PCL-NHS.  The branched PEI was reacted with mPEG-PCL-NHS to synthesize the ternary copolymers mPEG-PCL-g-PEI.  The structure of mPEG-PCL-g-PEI copolymers was characterized with Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC).  The mPEG-PCL-g-PEI/siRNA nanoparticles were prepared by complex coacervation, and the nanoparticles size and zeta potential were determined, separately.  The cytotoxicities of mPEG-PCL-g-PEI/siRNA nanoparticles and PEI/siRNA nanoparticles were compared through cells MTT assays in vitro.  The inhibition efficiencies of firefly luciferase gene expression by mPEG-PCL-g-PEI/ siRNA nanoparticle at various N/P ratios were investigated through cell transfection in vitro.  The experimental results suggested that the ternary (mPEG5k-PCL1.2k)1.4-g-PEI10k copolymers were successfully synthesized.  (mPEG5k-PCL1.2k)1.4-g-PEI10k could condense siRNA into nanoparticles (50−200 nm) with positive zeta potential.  MTT assay results showed that the cytotoxicity of (mPEG5k-PCL1.2k)1.4-g-PEI10k/siRNA nanoparticles was significantly lower than that of PEI10k/siRNA nanoparticles (P < 0.05).  The expression of firefly luciferase gene could be significantly down-regulated at a range of N/P ratio from 50 to 150 (P < 0.01), and maximally inhibited at the N/P ratio of 125.  The mPEG-PCL-g-PEI polymers could delivery siRNA into cells to inhibit the expression of target gene with very low cytotoxicity, which suggested that mPEG-PCL-g-PEI could serve as a new type of siRNA delivery vector.

     

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