Evaluation of generation 2 and 3 poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides targeting the epidermal growth factor receptor

被引:73
作者
Hollins, AJ
Benboubetra, M
Omidi, Y
Zinselmeyer, BH
Schatzlein, AG
Uchegbu, IF
Akhtar, S [1 ]
机构
[1] Univ Wales Coll Cardiff, Welsh Sch Pharm, Ctr Genome Based Therapeut, Cardiff CF10 3XF, S Glam, Wales
[2] Univ Setif, Fac Sci, Lab Appl Biochem, Setif 19000, Algeria
[3] Tabriz Univ Med Sci, Dept Pharmaceut, Tabriz 5166 15731, Iran
[4] Univ Strathclyde, Dept Pharmaceut Sci, Glasgow G4 0NR, Lanark, Scotland
[5] Univ Glasgow, Dept Med Oncol, Canc Res Campaign, Glasgow G61 1BD, Lanark, Scotland
关键词
A431; cells; antisense oligodeoxynucleotide; epidermal growth factor receptor; gene delivery; poly(propylenimine) dendrimer;
D O I
10.1023/B:PHAM.0000019300.04836.51
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Purpose. 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. Methods. 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. Results. G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximate to30 mug/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 4degreesC (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. Conclusions. 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.
引用
收藏
页码:458 / 466
页数:9
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