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目的研究聚乙二醇(PEG)不同接枝率对第五代聚酰胺-胺(polyanfido-amine,PAMAM G5)树状大分子体外毒性与细胞摄取的影响。方法合成4种不同接枝率的PEG-PAMAM G5,采用核磁共振氢谱(~1H-NMR)、红外光谱(FT-IR)、纳米粒度-电位分析仪进行结构鉴定和表征,溶血毒性和细胞毒性实验考察体外安全性,细胞摄取实验及激光共聚焦显微镜考察摄取情况及胞内定位。结果 4种PEG-PAMAM G5的PEG接枝率分别为7.8%,14.1%,20.3%和24.2%,粒径依次为(17.05±1.77)nm<(20.77±1.02)nm<(21.68±1.04)nm<(23.19±0.54)nm,Zeta电位由PAMAM G5的(25.57±1.37)mV降低至PEG_(31)-PAMAM G5的(9.27±0.40)mV。PAMAM G5经PEG修饰后,溶血毒性、细胞毒性和细胞摄取均显著降低(P<0.05),仍可被HBMEC细胞摄取并进入细胞核。结论 PEG修饰可显著降低PAMAM G5的体外细胞毒性,随着PEG接枝率的增大,Zeta电位降低,减毒效果越显著;经修饰后的PEG-PAMAM G5仍具有进入细胞核的能力。因此,PEG化树状大分子可作为基因或细胞核靶向药物的载体进行更深入的研究。
Aim To investigate the effects of different graft rates of polyethylene glycol (PEG) on the in vitro cytotoxicity and cytotoxicity of dendrimer of fifth generation polyamidoamine (PAMAM G5). Methods Four kinds of PEG-PAMAM G5 with different grafting degrees were synthesized and characterized by 1H-NMR, FT-IR and nano-particle size-potential analyzer. Toxicity tests in vitro safety, cellular uptake experiments and laser scanning confocal microscopy examination of intake and intracellular localization. Results The PEG grafting rates of the four PEG-PAMAM G5s were 7.8%, 14.1%, 20.3% and 24.2%, respectively. The diameters of the grafts were (17.05 ± 1.77) nm <(20.77 ± 1.02) nm <(21.68 ± 1.04) nm <(23.19 ± 0.54) nm, Zeta potential decreased from (25.57 ± 1.37) mV of PAMAM G5 to (9.27 ± 0.40) mV of PEG_ (31) -PAMAM G5. After PAMAM G5 was modified by PEG, the hemolytic toxicity, cytotoxicity and cell uptake were significantly decreased (P <0.05), and were still uptake by the HBMEC cells and into the nucleus. Conclusion PEG modification can significantly reduce the cytotoxicity of PAMAM G5 in vitro. With the increase of PEG grafting rate, the Zeta potential decreases and the attenuating effect becomes more remarkable. The modified PEG-PAMAM G5 still has the ability to enter the nucleus. Therefore, PEGylated dendrimer can be used as a carrier for gene or nuclear targeting drugs for further study.