TAT-LHRH-壳聚糖/siRNA纳米复合物与巨噬细胞的相互作用  

Interaction of TAT-LHRH conjugated chitosan/si RNA nano-polyplexes with macrophages

作　　者：邵楠[1] 董霞[1] 刘兰霞[1] 朱敦皖[1] 张海玲[1] 冷希岗[1] 

机构地区：[1]中国医学科学院.北京协和医学院生物医学工程研究所基因工程实验室,天津300192

出　　处：《生物医学工程与临床》2015年第4期339-344,共6页Biomedical Engineering and Clinical Medicine

基　　金：天津市自然科学基金重点项目(11JCZDJC20300);国家自然科学基金资助项目(81271693;31200674)

摘　　要：目的评价反式转录激活因子短肽-促黄体生成激素释放激素(TAT-LHRH)修饰的低分子质量壳聚糖(LMWC)作为siRNA载体的生物安全性。方法利用琥珀亚酰胺基-3-2-硫代吡啶-丙酸酯(SPDP)共价连接TAT-LHRH双功能肽及LMWC(相对分子质量5 000~8 000;脱乙酰化程度90%),合成TAT-LHRH-壳聚糖(TLC)载体,并与未修饰的LMWC分别和非编码siRNA(序列5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′)混合,合成TLC/siRNA纳米复合物和LMWC/siRNA纳米复合物。利用凝胶阻滞及光散射实验观察纳米粒的表征,并通过酶联免疫吸附分析(ELISA)、流式细胞术及体内巨噬细胞吞噬实验分析纳米复合物诱导小鼠单核巨噬细胞系RAW264.7所引起的细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-12(IL-12)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)的变化,以及对人巨噬细胞吞噬活性的影响。结果与未进行任何修饰的壳聚糖相比,TLC具有更好的siRNA结合能力(N/P比分别为1∶2、1∶1、2∶1、5∶1和10∶1)。以不同N/P(10∶1、20∶1)与siRNA形成纳米复合体后,其粒径分布于90~150 nm,zeta电位稳定在+2.7^+19.3 m V。含200 nmol/L siRNA的TLC/siRNA纳米复合物在24 h内未引起上述细胞因子的明显释放。体内与体外的巨噬细胞吞噬实验证明,相比未修饰壳聚糖/siRNA复合物而言,TLC/siRNA纳米复合物被巨噬细胞摄入量更高,但两者均未对吞噬活性和细胞因子生成产生明显影响。结论 TLC无明显免疫毒性,是一种有应用前景的siRNA载体。Objective To assess bio-safety of transactivator of transcription peptide luteinizing hormone-releasing hormone（TAT-LHRH） modified low molecular weight chitosan （LMWC） as siRNA delivery vector. Methods TAT-LHRH and LMWC （relative molecular weight was 5 000-6 000, and deacetylation degree was 90%） were linked by SPDP covalently to product TAT-LHRH conjugated chitosan（TLC）, and the unmodified LWMC was mixed with TLC at different concentration non-coding siRNA（sequence：5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′）, respectively, then obtained TLC/siRNA and LWMC/siRNA polyplexes. The characteristics of nanoparticles were observed by agarose gel retardation assay and light scattering, while analyzed the variation of cytokines [interleukin 1β（IL-1β）, IL-6, IL-12, tumor necrosis factorα（TNF-α） and IL-10] concentration after coincubating nano-particles with mouse mononuclear macrophage RAW264.7 by enzyme-linked immunosorbent assay （ELISA）, flow cytometry and macrophage phagocytosis test, which to detect the macrophage phagocytic activity influenced by nano-polyplex in vivo. Results The TLC showed stronger siRNA condensing power than unmodified chitosan（N/P ratios were 1∶2, 1∶1, 2∶1, 5∶1 and 10∶1, respectively）, and the size of TLC/siRNA nano-polyplexes（N/P ratios were 10∶1 and 20∶1） ranged 90-150 nm with zeta potential ranged＋2.7-＋19.3 mV. The TLC/siRNA nano-polyplexes containing 200 nmol/L siRNA were not induced the release of cytokines in 24-hour. Compared to unmodified chitosan/siRNA nano-polyplexes, the TLC/siRNA had higher intake of macrophages, but there was no obvious influence in phagocytic activity and cytokine. Conclusion It is demonstrated that TLC is a safe and promising siRNA vector without obvious toxicity.

关 键 词：siRNA运输 肝癌 反式转录激活因子短肽(TAT) 促黄体生成激素释放激素(LHRH) 壳聚糖 巨噬细胞 

分 类 号：R318.08[医药卫生—生物医学工程]