Yoo H, Sazani P, Juliano R L
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
Pharm Res. 1999 Dec;16(12):1799-804. doi: 10.1023/a:1018926605871.
To investigate the potential use of PAMAM dendrimers for the delivery of antisense oligonucleotides into cells under conditions that mimic the in vivo environment.
We used HeLa cells stably transfected with plasmid pLuc/705 which has a luciferase gene interrupted by a human beta-globin intron mutated at nucleotide 705, thus causing incorrect splicing. An antisense oligonucleotide overlapping the 705 splice site, when delivered effectively, corrects splicing and allows luciferase expression. The ability of dendrimers to deliver oligonucleotides to HeLa Luc/705 cells was evaluated in the absence or presence of serum.
PAMAM dendrimers formed stable complexes with oligonucleotides that had modest cytotoxicity and showed substantial delivery activity. The dose of the oligonucleotide, the charge ratio of oligonucleotide to dendrimer, and the size (generation) of the dendrimers were all critical variables for the antisense effect. The physical properties of dendrimer/oligonucleotide complexes were further investigated using sedimentation and gel electrophoresis methods. Effective oligonucleotide/generation 5 dendrimer complexes were macromolecular rather than particulate in nature, and were not sedimented at 100,000 RPM. Compared to other types of delivery agents, PAMAM dendrimers were more effective in delivering oligonucleotides into the nucleus of cells in the presence of serum proteins.
Our results suggest that PAMAM dendrimers form nonparticulate delivery complexes that function in the presence of serum proteins and thus may be suited for in vivo therapeutic applications.
研究聚酰胺 - 胺（PAMAM）树枝状大分子在模拟体内环境条件下将反义寡核苷酸递送至细胞中的潜在用途。
我们使用稳定转染了质粒pLuc/705的HeLa细胞，该质粒具有一个荧光素酶基因，该基因被在核苷酸705处突变的人β - 珠蛋白内含子打断，从而导致剪接错误。当有效递送时，与705剪接位点重叠的反义寡核苷酸可纠正剪接并允许荧光素酶表达。在有无血清的情况下评估树枝状大分子将寡核苷酸递送至HeLa Luc/705细胞的能力。
PAMAM树枝状大分子与寡核苷酸形成稳定的复合物，具有适度的细胞毒性并表现出显著的递送活性。寡核苷酸的剂量、寡核苷酸与树枝状大分子的电荷比以及树枝状大分子的大小（代数）都是反义效应的关键变量。使用沉降和凝胶电泳方法进一步研究了树枝状大分子/寡核苷酸复合物的物理性质。有效的寡核苷酸/第5代树枝状大分子复合物本质上是大分子而非颗粒状，并且在100,000转/分钟下不会沉降。与其他类型的递送剂相比，在存在血清蛋白的情况下，PAMAM树枝状大分子在将寡核苷酸递送至细胞核方面更有效。
我们的结果表明，PAMAM树枝状大分子形成非颗粒状递送复合物，在血清蛋白存在下起作用，因此可能适用于体内治疗应用。
