【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。更多还原

【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.更多还原