Kihara Fumihiro, Arima Hidetoshi, Tsutsumi Toshihito, Hirayama Fumitoshi, Uekama Kaneto
Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
Bioconjug Chem. 2002 Nov-Dec;13(6):1211-9. doi: 10.1021/bc025557d.
To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.
为提高新型非病毒载体——一种与α-环糊精偶联的聚酰胺-胺树枝状大分子（G2）（α-CDE偶联物（G2））的基因转移活性，我们制备了与不同代数树枝状大分子（G3和G4）偶联的α-CDE偶联物，并将它们的基因转移活性与α-CDE偶联物（G2）和新型转染试剂TransFast进行了比较。α-CDE偶联物（G2、G3和G4）与质粒DNA（pDNA）形成复合物，以电荷比依赖的方式改变pDNA复合物的zeta电位和粒径，并保护pDNA免受DNase I的降解，尽管它们在较高电荷比（载体/pDNA）下的差异较小。在NIH3T3和RAW264.7细胞中，α-CDE偶联物（G2、G3和G4）的基因转移活性高于相应的单独树枝状大分子。在这些CDE偶联物中，α-CDE偶联物（G3）具有优异的基因转移活性，在NIH3T3细胞中与TransFast相当。将与α-CDE偶联物（G3）形成的pDNA复合物应用于NIH3T3细胞后，pDNA的细胞内分布与单独使用树枝状大分子（G3）不同，尽管所有载体中pDNA的细胞结合情况几乎相当。α-CDE偶联物（G3）与荧光探针2-（对甲苯胺基）-萘-6-磺酸盐（TNS）强烈相互作用，表明该偶联物在转染后具有与生物膜成分如磷脂的包合能力。这些结果表明，α-CDE偶联物，特别是G3偶联物，可能是新型非病毒基因转移剂。
