Centre for Genome-based Therapeutics, Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3XF, Wales, UK.
Pharm Res. 2004 Mar;21(3):458-66. doi: 10.1023/B:PHAM.0000019300.04836.51.
To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells.
Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels.
G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery.
G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.
评估低代数、G2 和 G3 代聚（丙稀亚胺）树枝状聚合物作为表皮生长因子受体（EGFR）反义寡核苷酸（ODN）的细胞内传递载体的潜力，在 A431 表皮样癌细胞中。
采用台盼蓝排斥试验评价树枝状聚合物的细胞毒性。采用荧光激活细胞分类术分析研究荧光标记 ODN 的细胞摄取。采用荧光显微镜评估固定和活细胞中树枝状聚合物传递的 ODN 的细胞内命运。根据癌细胞生长、靶 EGFR 蛋白抑制和 mRNA 水平降低来评估反义 ODN 活性。
G2 树枝状聚合物（DAB-8）在 A431 细胞中的毒性低于 G3（DAB-16）树枝状聚合物，其 IC50 分别大于 175 和大约 30μg/ml。与液流胞吞作用的标记物相比，荧光标记 ODN：树枝状聚合物复合物的摄取增加了 100 倍，与游离 ODN 相比，在最佳树枝状聚合物：ODN（w/w）比为 5:1 时，摄取增加了 9 倍。在 4℃时，树枝状聚合物：ODN 复合物的摄取显著降低（p < 0.05）。活细胞荧光显微镜观察结果表明，树枝状聚合物：ODN 复合物的细胞内分布提示其为内吞作用摄取；相比之下，细胞固定导致人为的核定位。用抗 EGFR 反义 ODN：树枝状聚合物复合物处理 A431 细胞，可抑制细胞生长、蛋白和 mRNA 表达，达到与 Oligofectamine 介导的传递相当的水平。
G2 和 G3 代聚（丙稀亚胺）树枝状聚合物明显提高了 ODN 的传递和活性，因此可能代表了向培养细胞中传递 ODN 的通用试剂。
