花生五烯酸与透明质酸接枝物的合成及其抗肝癌活性

Synthesis of eicosapentaenoic acid and hyaluronic acid graft copolymer and its anti-hepatoma activity

合成花生五烯酸(EPA)与透明质酸(HA)耦合物,初步评价其体外抗肝癌活性。通过胱胺将花生五烯酸与透明质酸连接,合成了一种透明质酸-花生五烯酸接枝物(HA-EPA),利用核磁共振仪(^(1)H NMR)和傅里叶变换红外光谱仪(FT-IR)对其结构进行了表征,利用激光粒度及Zeta电位分析仪检测了其粒径与电位。采用MTT法检测了HA-EPA对肝癌细胞HepG2,Huh-7和正常肝细胞LX-2的体外抗细胞增殖作用。利用EdU染色与TUNEL染色法,考察了HA-EPA对HepG2细胞体外增殖与凋亡的影响。流式细胞术进一步验证了凋亡情况。通过迁移和侵袭实验考察了HA-EPA对HepG2细胞迁移和侵袭能力的影响。^(1)H NMR结果显示,HA-EPA被成功合成,且EPA在HA上的接枝率约(40±5)%;FT-IR结果进一步确证了HA-EPA的结构;粒径为(162.5±10.2)nm,电位为−(4.47±0.31)mV;MTT结果表明,随着药物处理时间延长,相同EPA含量下HA-EPA表现出优于EPA的对HepG2与Huh-7细胞活性抑制能力。当作用48 h,HA-EPA对正常肝细胞LX-2的毒性小于EPA。HepG2的24 h增殖、凋亡、迁移与侵袭实验结果显示,透明质酸的接枝提高了EPA抑制HepG2细胞增殖,促进凋亡,抑制迁移与侵袭的能力(P<0.001),说明HA的接枝可以明显增强EPA的肝癌抑制效果并起到一定的减毒作用。

In this study,the conjugate of eicosapentaenoic acid(EPA) and hyaluronic acid(HA) was synthesized and the anti-hepatoma activities in vitro were evaluated.The hyaluronic acid-eicosapentaenoic acid(HA-EPA)nanoparticle was synthesized by linking eicosapentaenoic acid with hyaluronic acid with cystamine.The structure of HA-EPA was characterized by nuclear magnetic resonance(1 H NMR) and Fourier transform infrared spectroscopy(FT-IR).Laser particle sizer and Zeta potential analyzer were used to detect the size and potential of HAEPA.MTT assay was used to detect the anti-proliferative effect of HA-EPA on HepG2,Huh-7 and LX-2 cells in vitro.The effects of HA-EPA nanoparticles on the proliferation and apoptosis of HepG2 cells in vitro were investigated by EdU staining and TUNEL staining.The apoptosis was further confirmed by flow cytometry.The effect of HA-EPA nanoparticles on the migration and invasion of HepG2 cells was demonstrated by transwell and invasion experiments.The results of1 H NMR showed that HA-EPA was successfully synthesized,and the grafting rate of EPA on HA was(40 ± 5) %.The structure of HA-EPA was further confirmed by FT-IR.The particle size was(162.5 ± 10.2) nm,and the potential was-(4.47 ± 0.31) mV.MTT results showed that,with the prolongation of drug treatment time,HA-EPA showed a better inhibitory effect on the activity of HepG2 and Huh-7 cells than EPA under the same EPA content.After treated for 48 hours,the toxicity of HA-EPA to LX-2 cells was less than that of EPA.The results of 24-hour proliferation,apoptosis,migration and invasion of HepG2 showed that,the graft of hyaluronic acid improved the ability of EPA to inhibit proliferation,promote apoptosis,migration and invasion of HepG2 cells(P < 0.001),indicating that grafting of HA can significantly enhance the inhibitory effect of EPA on liver cancer with some role in reducing toxicity.