High functionalization of dendrimer/cyclodextrin conjugate as a novel siRNA carrier

我们以前报道了星爆聚酰胺-胺树枝状聚合物（dendrimer，generation 3，G3）与α-环糊精（α-CDE）缀合物作为新型基因递送载体的潜在用途。本研究在体外和体内研究了α-CDE及其糖修饰的α-CDE作为小干扰RNA（siRNA）和短发夹RNA表达载体（shpDNA）的细胞特异性载体的潜力。在稳定表达pGL 3荧光素酶的NIH 3 T3细胞中，具有cc-CDE的siRNA复合物的RNAi效果优于具有市售siRNA载体的复合物的RNAi效果。此外，siRNA与a-CDE的复合物显示出可忽略的细胞毒性，直至更高的电荷比。然而，我们发现a-CDE由于低RNAi效应而不具有作为shpDNA载体的潜力。当将含有siRNA与α-CDE的二元复合物的溶液注射到携带稳定表达GL 3荧光素酶基因的Colon-26细胞的小鼠的肿瘤组织中时，提供了足够的RNAi效应，尽管LipofectamineTM 2000与siRNA的复合物显示出脱靶效应。因此，发现α-CDE通过与siRNA复合提供序列特异性基因沉默效应。另一方面，我们制备了甘露糖基化α-CDE（M-α-CDE）和乳糖基化α-CDE（L-α-CDE），分别将siRNA和shpDNA特异性地递送到抗原呈递细胞和肝细胞。然而，我们发现L-α-CDE能够与肝细胞上表达的去唾液酸糖蛋白受体结合，而M-a-CDE不能与抗原呈递细胞上表达的甘露糖受体结合。L-α-CDE和M-α-CDE的细胞毒性可忽略不计。这些结果表明L-α-CDE具有作为肝细胞特异性siRNA和shpDNA载体的潜力。

We previously reported the potential use of starburst polyamidoamine dendrimer (dendrimer, generation 3, G3)conjugate with a-cyclodextrin (α-CDE) as a novel gene delivery carrier. In the present study, the potential of α-CDE and their sugar-appended α-CDEs as novel and cell-specific carriers of small interfering RNA(siRNA) and short hairpin RNA-expressing vector(shpDNA)was evaluated in vitro and in vivo. The RNAi effects of the siRNA complex with cc-CDE were preferable to those of the complexes with commercially-available siRNA carriers in NIH3T3 cells stably expressing pGL3 luciferase. In addition, the siRNA complex with a-CDE showed negligible cytotoxicity up to higher charge ratios. However, we found that a-CDE did not have the potential as shpDNA carriers because of low RNAi effect. When the solution containing the binary complex of siRNA with α-CDE was injected into tumor tissues of mice bearing Colon-26 cells stably expressing the GL3 luciferase gene, the sufficient RNAi effects were provided, although the complex of Lipofectamine^TM2000 with siRNA showed the off-target effect. Therefore, α-CDE was found to provide the sequence-specific gene silencing effect by the complexation with siRNA. On the other hand, we prepared mannosylated α-CDE(M-α-CDE)and lactosylated α-CDE(L-α-CDE)to delivery siRNA and shpDNA specifically to antigen-presenting cells and hepatocytes, respectively. However, we found that L-α-CDE was able to bind to asialoglycoprotein receptor expressing on hepatocytes, although M-a-CDE could not bind to mannose receptor expressing on antigen-presenting cells. Both L-α-CDE and M-α-CDE had negligible cytotoxicity. These results suggest that L-α-CDE has the potential for hepatocyte-specific siRNA and shpDNA carriers.